Substituted 6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepines as anti-proliferative agents

ABSTRACT

The invention relates to pyrridinobenzodiazepines (PDDs) comprising three fused 6-7-6-membered rings linked to aromatic groups, and pharmaceutically acceptable salts thereof, which are useful as medicaments, in particular as anti-proliferative agents. PDDs may be represented by formula (I) and pharmaceutically acceptable salts thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International patentapplication number PCT/GB2016/052565, filed 19 Aug. 2016, (currentlypublished). International patent application number PCT/GB2016/05265cites the priority of Great Britain patent application numberGB1514928.9, filed 21 Aug. 2015, (abandoned).

FIELD OF THE INVENTION

The invention relates to pyrridinobenzodiazepines (PDDs) comprisingthree fused 6-7-6-membered rings. In particular it relates to compoundscomprising a PDD group linked via the A-ring to aromatic groups, and topharmaceutically acceptable salts thereof, which are useful asmedicaments, in particular as anti-proliferative agents.

BACKGROUND TO THE INVENTION

Pyrridinobenzodiazepines (PDDs) are related structures topyrrolobenzodiazepines (PBDs). The pyrrolobenzodiazepines (PBDs) are agroup of compounds some of which have been shown to besequence-selective DNA minor-groove binding agents. The PBDs wereoriginally discovered in Streptomyces species (1-5). They are tricyclicin nature, and are comprised of fused 6-7-5-membered rings that comprisean anthranilate (A ring), a diazepine (B ring) and a pyrrolidine (Cring) (3). They are characterized by an electrophilic N10=C11 iminegroup (as shown below) or the hydrated equivalent, a carbinolamine[NH—CH(OH)], or a carbinolamine alkyl ether ([NH—CH(OR, where R=alkyl)]which can form a covalent bond to a C2-amino group of guanine in DNA toform a DNA adduct (6).

The natural products interact in the minor groove of the DNA helix withexcellent fit (i.e., good “isohelicity”) due to a right-handedlongitudinal twist induced by a chiral C11a-position which has the(S)—configuration (6). The DNA adduct has been reported to inhibit anumber of biological processes including the binding of transcriptionfactors (7-9) and the function of enzymes such as endonucleases (10, 11)and RNA polymerase (12). PBD monomers (e.g., anthramycin) have beenshown by footprinting (6), NMR (13, 14), molecular modeling (15) andX-ray crystallography (16) to span three base pairs and to have athermodynamic preference for the sequence 5′-Pu-G-Pu-3′ (wherePu=purine, and G is the reacting guanine) (17) and a kinetic preferencefor Py-5-Py (where Py=Pyrimidine).

PBDs are thought to interact with DNA by first locating at a low-energybinding sequence (i.e., a 5′-Pu-G-Pu-3′ triplet) through Van der Waals,hydrogen bonding and electrostatic interactions (7). Then, once inplace, a nucleophilic attack by the exocyclic C2-amino group of thecentral guanine occurs to form the covalent adduct (7). Once bound, thePBD remains anchored in the DNA minor groove, avoiding DNA repair bycausing negligible distortion of the DNA helix (16). The ability of PBDsto form an adduct in the minor groove and crosslink DNA enables them tointerfere with DNA processing and, hence, their potential for use asantiproliferative agents.

A number of monomeric PBD structures have been isolated fromStreptomyces species, including anthramycin (18) the first PBD,tomamycin (19), and more recently usabamycin (20) from a marine sedimentStreptomyces species in a marine sediment. This has led to thedevelopment of a large range of synthetic analogues which have beenreviewed (1, 21). More recently, a number of monomeric PBD structuresthat are linked through their C8 position to pyrroles and imidazoleshave been reported WO 2007/039752, WO 2013/164593 (22-27).

WO 2010/091150 discloses a dimer of a 6-7-6 ring system linked via theirA-rings. WO 2015/028850 discloses 6-7-5 ring system PBD dimers that arelinked via phosphine oxide containing linkers attached to their aromaticA-rings. In addition, WO 2015/028850 discloses a dimer compoundcontaining a 6-7-6 ring system linked via the key phosphine oxidecontaining linkers.

Various PBDs have been shown to act as cytotoxic agents in vitro, forexample, WO 00/12508, WO 2004/087711, and as anti-tumour in vivo inanimal tumour models, for example, WO 2011/117882, WO 2013/164593.Furthermore, the C8/C8′-linked PBD dimer SJG-136 (28, 29) has completedPhase I clinical trials for leukaemia and ovarian cancer (30) and hasshown sufficient therapeutic benefit to progress to Phase II studies.

However, results from a Phase I clinical evaluation of SJG-136 revealedthat the drug produced several adverse effects including lower-limbedema and fatigue (31).

Thus, there exists a need for further compounds related to PBDs that aretherapeutically active for treating a variety of proliferative diseases.

The present application reports pyrridinobenzodiazepines (PDDs), whichare related to PBDs but contain an expanded 6-membered C-ring ascompared to the 5-membered C-ring of PBDs. The inventors have discoveredthat PDD conjugates provide properties, such as cytoxicity and DNAbinding, that results in effective compounds.

The present invention seeks to overcome problem(s) associated with theprior art.

SUMMARY OF THE INVENTION

The present invention provides a compound of formula (I):

and salts and solvates thereof,

wherein;

the dotted lines indicates the optional presence of a double bondbetween one or more of C1 and C2, C2 and C3, and C3 and C4;

R₁ is selected from R₇, ═CH₂, ═CH—(CH₂)_(m)—CH₃, ═O, (CH₂)_(m)—OR₇,(CH₂)_(m)—CO₂R₇, (CH₂)_(m)—NR₇R₈, O—(CH₂)_(n)—NR₇R₈, NH—C(O)—R₇,O—(CH₂)_(n)—NH—C(O)—R₇, O—(CH₂)_(n)—C(O)—NH—R₇, (CH₂)_(m)—SO₂R₇,O—SO₂R₇, (CH₂)_(m)—C(O)R₇ and (CH₂)_(m)—C(O)NR₇R₈;

R₂ is selected from R₉, ═CH₂, ═CH—(CH₂)_(r)—CH₃, ═O, (CH₂)_(r)—OR₉,(CH₂)_(r)—CO₂R₉, (CH₂)_(r)—NR₉R₁₀, O—(CH₂)_(s)—NR₉R₁₀, NH—C(O)—R₉,O—(CH₂)_(s)—NH—C(O)—R₉, O—(CH₂)_(s)—C(O)—NH—R₉, (CH₂)_(r)—SO₂R₉,O—SO₂R₉, (CH₂)_(r)—COR₉ and (CH₂)_(r)—C(O)NR₉R₁₀;

R₃ is selected from H, C₁₋₁₂ alkyl and CH₂Ph;

R₄ is selected from phenyl and C₅₋₉ heteroaryl groups optionallysubstituted with up to three optional substituent groups selected fromOH, C₁₋₆ alkyl, OC₁₋₆ alkyl, (CH₂)_(j)—CO₂R₁₁, O—(CH₂)_(k)—NR₁₁R₁₂,(CH₂)_(j)—NR₁₁R₁₂, C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂; C(═O)—NH—R₂₄

and C(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂; with the proviso that theoptionally substituted C₅₋₉ heteroaryl is not indolyl;

R₁₉ is selected from H and (CH₂)_(t)—NR₂₀R₂₁;

Y₁ is N or CH;

Y₂ is N or CH; and wherein at least one of Y₁ and Y₂ is CH;

p is 0 or 1;

j, m, r and t are independently selected from an integer from 0 to 6;

k, n and s are independently selected from an integer from 1 to 6;

X₁ is selected from O, S, NR₁₃, CR₁₃R₁₄, CR₁₃R₁₄O, C(═O), C(═O)NR₁₃,NR₁₃C(═O), O—C(O) and C(O)—O;

L is selected from an amino acid, a peptide chain having from 2 to 6amino acids, an alkylene chain containing from 1 to 12 carbon atomswhich may contain one or more carbon-carbon double or triple bonds, aparaformaldehyde chain—(OCH₂)₁₋₁₂—, a polyethylene glycolchain—(OCH₂CH₂)₁₋₆—, which chains may be interrupted by one or more ofO, S and/or NH groups and/or C₃₋₉ heteroarylene and/or phenylene;

X₂ is selected from O, S, NR₁₅, CR₁₅R₁₆, CR₁₅R₁₆O, C(═O), C(═O)NR₁₅,NR₁₅C(═O), O—C(O) and C(O)—O or is absent;

q is selected from 0, 1, 2, 3, 4, 5 and 6;

A is selected from:

-   -   for each A1 group one of Y₃ and Y₄ is independently selected        from N—R₁₇, S and O; and the other of Y₃ and Y₄ is CH; and Y₅ is        independently selected from CH, N, S and COH; and    -   for each A2 group one of Y₆ and Y₇ is independently selected        from N and CH; and the other of Y₆ and Y₇ is CH;

R₇ and R₉ are independently selected from H, C₁₋₁₂ alkyl, C₅₋₉heteroaryl, C₆₋₁₅ heteroarylalkyl, phenyl and C₇₋₁₂ aralkyl groups;wherein the heteroaryl, heteroarylalkyl, phenyl and aralkyl groups areoptionally substituted with up to three optional substituent groupsselected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl;

R₂₄ is a phenyl optionally substituted with up to three optionalsubstituent groups selected from OH, C₁₋₆ alkyl, OC₁₋₆ alkyl,(CH₂)_(j)—CO₂R₁₁, O—(CH₂)_(k)—NR₁₁R₁₂, (CH₂)_(j)—NR₁₁R₁₂,C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂ and C(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂;

R₈, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₂₀ and R₂₁ areindependently selected from H and C₁₋₆ alkyl;

and either:

-   -   (i) R₅ and R₆ together form a double bond;    -   (ii) R₅ is H and R₆ is OH; or    -   (iii) R₅ is H and R₆ is OC₁₋₆ alkyl;

with the proviso that when p is 0 and A is A1, then:

-   -   (a) for at least one A1 group one of Y₃ and Y₄ is selected from        S and O; or    -   (b) for at least one A1 group Y₅ is S; or    -   (c) R₄ is not pyrrolyl, imidazolyl, optionally substituted        pyrrolyl or optionally substituted imidazolyl.

The present invention provides a compound of formula (I):

and salts and solvates thereof,

wherein;

the dotted lines indicates the optional presence of a double bondbetween one or more of C1 and C2, C2 and C3, and C3 and C4;

R₁ is selected from R₇, ═CH₂, ═CH—(CH₂)_(m)—CH₃, ═O, (CH₂)_(m)—OR₇,(CH₂)_(m)—CO₂R₇, (CH₂)_(m)—NR₇R₈, O—(CH₂)_(n)—NR₇R₈, NH—C(O)—R₇,O—(CH₂)_(n)—NH—C(O)—R₇, O—(CH₂)_(n)—C(O)—NH—R₇, (CH₂)_(m)—SO₂R₇,O—SO₂R₇, (CH₂)_(m)—C(O)R₇ and (CH₂)_(m)—C(O)NR₇R₈;

R₂ is selected from R₉, ═CH₂, ═CH—(CH₂)_(r)—CH₃, ═O, (CH₂)_(r)—OR₉,(CH₂)_(r)—CO₂R₉, (CH₂)_(r)—NR₉R₁₀, O—(CH₂)_(s)—NR₉R₁₀, NH—C(O)—R₉,O—(CH₂)_(s)—NH—C(O)—R₉, O—(CH₂)_(s)—C(O)—NH—R₉, (CH₂)_(r)—SO₂R₉,O—SO₂R₉, (CH₂)_(r)—COR₉ and (CH₂)_(r)—C(O)NR₉R₁₀;

R₃ is selected from H, C₁₋₁₂ alkyl and CH₂Ph;

R₄ is selected from phenyl and C₅₋₉ heteroaryl groups optionallysubstituted with up to three optional substituent groups selected fromOH, C₁₋₆ alkyl, OC₁₋₆ alkyl, (CH₂)_(j)—CO₂R₁₁, O—(CH₂)_(k)—NR₁₁R₁₂,(CH₂)_(j)—NR₁₁R₁₂, C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂;C(═O)—NH—C₆H₄—(CH₂)_(j)—R₁₈ and C(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂; withthe proviso that the optionally substituted C₅₋₉ heteroaryl is notindolyl;

R₁₉ is selected from H and (CH₂)_(t)—NR₂₀R₂₁;

Y₁ is N or CH;

Y₂ is N or CH; and wherein at least one of Y₁ and Y₂ is CH;

p is 0 or 1;

j, m, r and t are independently selected from an integer from 0 to 6;

k, n and s are independently selected from an integer from 1 to 6;

X₁ is selected from O, S, NR₁₃, CR₁₃R₁₄, CR₁₃R₁₄O, C(═O), C(═O)NR₁₃,NR₁₃C(═O), O—C(O) and C(O)—O;

L is selected from an amino acid, a peptide chain having from 2 to 6amino acids, an alkylene chain containing from 1 to 12 carbon atomswhich may contain one or more carbon-carbon double or triple bonds, aparaformaldehyde chain —(OCH₂)₁₋₁₂—, a polyethylene glycol chain—(OCH₂CH₂)₁₋₆—, which chains may be interrupted by one or to more of O,S and/or NH groups and/or C₃₋₉ heteroarylene and/or phenylene;

X₂ is selected from O, S, NR₁₅, CR₁₅R₁₆, CR₁₅R₁₆O, C(═O), C(═O)NR₁₅,NR₁₅C(═O), O—C(O) and C(O)—O or is absent;

q is selected from 0, 1, 2, 3, 4, 5 and 6;

A is selected from:

-   -   for each A1 group one of Y₃ and Y₄ is independently selected        from N—R₁₇, S and O; and the other of Y₃ and Y₄ is CH; and Y₅ is        independently selected from CH, N, S and COH; and    -   for each A2 group one of Y₆ and Y₇ is independently selected        from N and CH; and the other of Y₆ and Y₇ is CH;

R₇ and R₉ are independently selected from H, C₁₋₁₂ alkyl, C₅₋₉heteroaryl, C₆₋₁₅ heteroarylalkyl, phenyl and C₇₋₁₂ aralkyl groups;wherein the heteroaryl, heteroarylalkyl, phenyl and aralkyl groups areoptionally substituted with up to three optional substituent groupsselected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl; R₁₈ is selected from H,CO₂R₁₁ and NR₁₁R₁₂;

R₈, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₂₀ and R₂₁ areindependently selected from H and C₁₋₆ alkyl;

and either:

-   -   (i) R₅ and R₆ together form a double bond;    -   (ii) R₅ is H and R₆ is OH; or    -   (iii) R₅ is H and R₆ is OC₁₋₆ alkyl;

with the proviso that when p is 0 and A is A1, then:

-   -   (a) for at least one A1 group one of Y₃ and Y₄ is selected from        S and O; or    -   (b) for at least one A1 group Y₅ is S; or    -   (c) R₄ is not pyrrolyl, imidazolyl, optionally substituted        pyrrolyl or optionally substituted imidazolyl.

In a further aspect, In a further aspect, there is provided a compoundof formula (I) and salts and solvates thereof for use in a method oftherapy.

In a further aspect, there is provided a compound of formula (I) andsalts and solvates thereof for use as a medicament.

In a further aspect, there is provided a compound of formula (I) andsalts and solvates thereof for use in the treatment of a proliferativedisease.

In a further aspect, there is provided a pharmaceutical compositioncomprising a compound of formula (I) and salts and solvates thereof anda pharmaceutically acceptable excipient, carrier or diluent.

In a further aspect, the present invention provides the use of acompound of formula (I) and salts and solvates thereof in themanufacture of a medicament for treating a proliferative disease.

In a further aspect, the present invention provides a method oftreatment of a patient suffering from a proliferative disease,comprising administering to said patient a therapeutically effectiveamount of a compound of formula (I) and salts and solvates thereof or apharmaceutical composition of the present invention.

In a further aspect, the compound of formula (I) and salts and solvatesthereof may be administered alone or in combination with othertreatments, either simultaneously or sequentially depending upon thecondition to be treated.

The pharmaceutical composition of the present invention may furthercomprise one or more (e.g. two, three or four) further active agents.

In a further aspect of the compound of formula (I) and salts andsolvates thereof, the following proviso applies: i) L is a peptide chainhaving from 2 to 6 amino acids, an alkylene chain containing from 1 to12 carbon atoms which may contain one or more carbon-carbon double ortriple bonds, a paraformaldehyde chain, a polyethylene glycol chain,which chains are interrupted by one or more of C₅₋₉ heteroarylene,phenylene or combinations of the foregoing; and/or ii) p is 1; and/oriii) q is 1, 2, 3, 4, 5 or 6.

In a further aspect, the compound of formula (I) and salts and solvatesthereof, may be linked, either directly or indirectly, to a targetingagent (e.g., antibody, antibody fragment, hormone, etc.) to provide atargeted conjugate. In a further aspect, the compound of formula (I) andsalts and solvates thereof, may contain a linker group, wherein thetargeting agent is attached to the compound of formula (I) and salts andsolvates thereof, through the linker group. The target conjugates of thepresent disclosure may contain one or multiple compounds of formula (I)(or salts and solvates thereof). A variety of target conjugates areknown in the art and may be used with a compound of formula (I) andsalts and solvates thereof. For example, in a particular aspect thetarget conjugate is an antibody-drug conjugate, wherein one or morecompounds of formula (I) are linked, directly or indirectly, to theantibody. Therefore, the compound of formula (I) and salts and solvatesthereof, may be used as a payload on a targeted conjugate.

DEFINITIONS

The following abbreviations are used throughout the specification: Acacetyl; Alloc allyloxycarbonyl; BAIBbis(acetoxy)iodobenzene/(diacetoxyiodo)benzene; Boc tert-butoxycarbonyl;BPDs benzopyrridodiazecines; CBz benzyloxycarbonyl; DBU1,8-diazabicyclo[5.4.0]undec-7-ene; DHP dihydropyran; DMAP4-dimethylaminopyridine; DMF dimethylformamide; DMSO dimethylsulfoxide;EDCl 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; Et ethyl; Et₂Odiethyl ether; EtOAc ethyl acetate; EtOH ethanol; HATU(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate); HMDST hexamethyldisilathiane; iBuiso-butyl; KOtBu potassium t-butoxide; L-Selectride Lithiumtri-sec-butyl(hydride)borate; Me methyl; MeOH methanol; PBDspyrrolo[2,1-c][1,4]benzo-diazepines; PDDs pyrridinobenzodiazepines; PIFAphenyliodine (III) bis[trifluoroacetate]; Ph phenyl; p-TSA/PTSAp-Toluenesulfonic acid; Pyr pyridine; TBAF tetrabutylammonium fluoride;TBS—Cl/TBDMSCl tert-butyldimethylsilyl chloride; TEA triethylamine;TEMPO (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl; TFA trifluoroaceticacid; THF tetrahydrofuran; THP tetrahydropyranyl; Troc2,2,2-Trichloroethyl carbonate and Ts (tosylate) p-toluene sulfonicacid.

“Substituted”, when used in connection with a chemical substituent ormoiety (e.g., an alkyl group), means that one or more hydrogen atoms ofthe substituent or moiety have been replaced with one or morenon-hydrogen atoms or groups, provided that valence requirements are metand that a chemically stable compound results from the substitution.

“Optionally substituted” refers to a parent group which may beunsubstituted or which may be substituted with one or more substituents.Suitably, unless otherwise specified, when optional substituents arepresent the optional substituted parent group comprises from one tothree optional substituents. Where a group may be “optionallysubstituted with up to three groups”, this means that the group may besubstituted with 0, 1, 2 or 3 of the optional substituents. Where agroup may be “optionally substituted with one or two optionalsubstituents”, this means that the group may be substituted with 0, 1 or2 of the optional substituents. Suitably groups may be optionallysubstituted with 0 or 1 optional substituents.

“Independently selected” is used in the context of statement that, forexample, “R₁ and R₂ are independently selected from H, C₁₋₁₂ alkyl,phenyl, . . . ” and means that each instance of the functional group,e.g. R₁, is selected from the listed options independently of any otherinstance of R₁ or R₂ in the compound. Hence, for example, a C₁₋₁₂ alkylmay be selected for the first instance of R₁ in the compound; a phenylgroup may be selected for the next instance of R₁ in the compound; and Hmay be selected for the first instance of R₂ in the compound.

C₁₋₁₂ alkyl: refers to straight chain and branched saturated hydrocarbongroups, generally having from 1 to 12 carbon atoms; more suitably C₁₋₇alkyl; more suitably C₁₋₆ alkyl. Examples of alkyl groups includemethyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl,pent-1-yl, pent-2-yl, pent-3-yl, 3-methylbut-1-yl, 3-methylbut-2-yl,2-methylbut-2-yl, 2,2,2-trimethyleth-1-yl, n-hexyl, n-heptyl, and thelike.

“Alkylene” refers to a divalent radical derived from an alkane which maybe a straight chain or branched, as exemplified by —CH₂CH₂CH₂CH₂—.

“Aryl”: refers to fully unsaturated monocyclic, bicyclic and polycyclicaromatic hydrocarbons having at least one aromatic ring and having aspecified number of carbon atoms that comprise their ring members (e.g.,C₆₋₁₄ aryl refers to an aryl group having 6 to 14 carbon atoms as ringmembers). The aryl group may be attached to a parent group or to asubstrate at any ring atom and may include one or more non-hydrogensubstituents unless such attachment or substitution would violatevalence requirements. Examples of aryl groups include phenyl.

“C₇₋₁₂ aralkyl” refers to an arylalkyl group having 7 to 12 carbon atomsand comprising an alkyl group substituted with an aryl group. Suitablythe alkyl group is a C₁₋₆ alkyl group and the aryl group is phenyl.Examples of C₇₋₁₂ aralkyl include benzyl and phenethyl. In some casesthe C₇₋₁₂ aralkyl group may be optionally substituted and an example ofan optionally substituted C₇₋₁₂ aralkyl group is 4-methoxylbenzyl.

“C₅₋₉ heteroaryl”: refers to unsaturated monocyclic or bicyclic aromaticgroups comprising from 5 to 9 ring atoms, whether carbon or heteroatoms,of which from 1 to 5 are ring heteroatoms. Suitably, any monocyclicheteroaryl ring has from 5 to 6 ring atoms and from 1 to 3 ringheteroatoms. Suitably each ring heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur. The bicyclic rings include fused ringsystems and, in particular, include bicyclic groups in which amonocyclic heterocycle comprising 5 ring atoms is fused to a benzenering. The heteroaryl group may be attached to a parent group or to asubstrate at any ring atom and may include one or more non-hydrogensubstituents unless such attachment or substitution would violatevalence requirements or result in a chemically unstable compound.

Examples of monocyclic heteroaryl groups include, but are not limitedto, those derived from:

N₁: pyrrole, pyridine;

O₁: furan;

S₁: thiophene;

N₁O₁: oxazole, isoxazole, isoxazine;

N₂O₁: oxadiazole (e.g. 1-oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl,1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl);

N₃O₁: oxatriazole;

N₁S₁: thiazole, isothiazole;

N₂: imidazole, pyrazole, pyridazine, pyrimidine, pyrazine;

N₃: triazole, triazine; and,

N₄: tetrazole.

Examples of heteroaryl which comprise fused rings, include, but are notlimited to, those derived from:

O₁: benzofuran, isobenzofuran;

N₁: indole, isoindole, indolizine, isoindoline;

S₁: benzothiofuran;

N₁O₁: benzoxazole, benzisoxazole;

N₁S₁: benzothiazole;

N₂: benzimidazole, indazole;

O₂: benzodioxole;

N₂O₁: benzofurazan;

N₂S₁: benzothiadiazole;

N₃: benzotriazole; and

N₄: purine (e.g., adenine, guanine), pteridine;

“Heteroarylene” refers to a divalent radical derived from a heteroarylgroup, as exemplified by pyridinylene —(C₅H₃N)—.

“C₆₋₁₅ heteroarylalkyl” refers to an alkyl group substituted with aheteroaryl group. Suitably the alkyl is a C₁₋₆ alkyl group and theheteroaryl group is C₅₋₉ heteroaryl as defined above. Examples of C₆₋₁₅heteroarylalkyl groups include pyrrol-2-ylmethyl, pyrrol-3-ylmethyl,pyrrol-4-ylmethyl, pyrrol-3-ylethyl, pyrrol-4-ylethyl,imidazol-2-ylmethyl, imidazol-4-ylmethyl, imidazol-4-ylethyl,thiophen-3-ylmethyl, furan-3-ylmethyl, pyridin-2-ylmethyl,pyridin-2-ylethyl, thiazol-2-ylmethyl, thiazol-4-ylmethyl,thiazol-2-ylethyl, pyrimidin-2-ylpropyl, and the like.

Nitrogen Protecting Groups

Nitrogen protecting groups are well known in the art. Preferred nitrogenprotecting groups are carbamate protecting groups that have the generalformula:

A large number of possible carbamate nitrogen protecting groups arelisted on pages 706 to 771 of Wuts, P. G. M. and Greene, T. W.,Protective Groups in Organic Synthesis, 4^(th) Edition,Wiley-Interscience, 2007, and in P. Kocienski, Protective Groups, 3rdEdition (2005) which are incorporated herein by reference.

Particularly preferred protecting groups include Alloc(allyloxycarbonyl), Troc (2,2,2-Trichloroethyl carbonate), Teoc[2-(Trimethylsilyl)ethoxycarbony], BOC (tert-butyloxycarbonyl), Doc(2,4-dimethylpent-3-yloxycarbonyl), Hoc (cyclohexyloxy-carbonyl), TcBOC(2,2,2-trichloro-tert-butyloxycarbonyl), Fmoc(9-fluorenylmethyloxycarbonyl), 1-Adoc (1-Adamantyloxycarbonyl) and2-Adoc (2-adamantyloxycarbonyl).

Hydroxyl Protecting Groups

Hydroxyl protecting groups are well known in the art, a large number ofsuitable groups are described on pages 16 to 366 of Wuts, P. G. M. andGreene, T. W., Protective Groups in Organic Synthesis, 4^(th) Edition,Wiley-Interscience, 2007, and in P. Kocienski, Protective Groups, 3rdEdition (2005) which are incorporated herein by reference.

Classes of particular interest include silyl ethers, methyl ethers,alkyl ethers, benzyl ethers, esters, benzoates, carbonates, andsulfonates.

Particularly preferred protecting groups include THP (tetrahydropyranylether).

“Compound of formula (I) and salts and solvates thereof” refers to thecompounds of formula (I); salts of compounds of formula (I); solvates ofcompounds of formula (I); and solvates of salts of compounds of formula(I).

“Drug”, “drug substance”, “active pharmaceutical ingredient”, and thelike, refer to a compound (e.g., compounds of formula (I) and compoundsspecifically named above) that may be used for treating a subject inneed of treatment.

“Excipient” refers to any substance that may influence thebioavailability of a drug, but is otherwise pharmacologically inactive.

“Pharmaceutically acceptable” substances refers to those substanceswhich are within the scope of sound medical judgment suitable for use incontact with the tissues of subjects without undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit-to-risk ratio, and effective for their intended use.

“Pharmaceutical composition” refers to the combination of one or moredrug substances and one or more excipients.

The term “subject” as used herein refers to a human or non-human mammal.Examples of non-human mammals include livestock animals such as sheep,horses, cows, pigs, goats, rabbits and deer; and companion animals suchas cats, dogs, rodents, and horses.

“Therapeutically effective amount” of a drug refers to the quantity ofthe drug or composition that is effective in treating a subject and thusproducing the desired therapeutic, ameliorative, inhibitory orpreventative effect. The therapeutically effective amount may depend onthe weight and age of the subject and the route of administration, amongother things.

“Treating” refers to reversing, alleviating, inhibiting the progress of,or preventing a disorder, disease or condition to which such termapplies, or to reversing, alleviating, inhibiting the progress of, orpreventing one or more symptoms of such disorder, disease or condition.

“Treatment” refers to the act of “treating”, as defined immediatelyabove.

As used herein the term “comprising” means “including at least in partof” and is meant to be inclusive or open ended. When interpreting eachstatement in this specification that includes the term “comprising”,features, elements and/or steps other than that or those prefaced by theterm may also be present. Related terms such as “comprise” and“comprises” are to be interpreted in the same manner.

R₁

R₁ is selected from R₇, ═CH₂, ═CH—(CH₂)_(m)—CH₃, ═O, (CH₂)_(m)—OR₇,(CH₂)_(m)—CO₂R₇, (CH₂)_(m)—NR₇R₈, O—(CH₂)_(n)—NR₇R₈, NH—C(O)—R₇,O—(CH₂)_(n)—NH—C(O)—R₇, O—(CH₂)_(n)—C(O)—NH—R₇, (CH₂)_(m)—SO₂R₇,O—SO₂R₇, (CH₂)_(m)—C(O)R₇ and (CH₂)_(m)—C(O)NR₇R₈. For the options whereR₁ is selected from ═CH₂, ═CH—(CH₂)_(m)—CH₃ and ═O, the carbon of theC-ring to which it is attached cannot have an optional double bond inorder for the valence requirements of the molecule to be met. Forexample, if R₁ is ═CH₂, and is positioned at the C1 position of theC-ring adjacent to the fused carbon of the C-ring, and R₂ is H then theresulting compound of formula (I) may be represented as:

Suitably R₁ is selected from R₇, (CH₂)_(m)—OR₇, (CH₂)_(m)—CO₂R₇,(CH₂)_(m)—NR₇R₈, O—(CH₂)_(n)—NR₇R₈, NH—C(O)—R₇, O—(CH₂)_(n)—NH—C(O)—R₇,O—(CH₂)_(n)—C(O)—NH—R₇, (CH₂)_(m)—SO₂R₇, O—SO₂R₇, (CH₂)_(m)—C(O)R₇ and(CH₂)_(m)—C(O)NR₇R₈.

Suitably R₁ is selected from R₇, (CH₂)_(m)—OR₇, (CH₂)_(m)—CO₂R₇,(CH₂)_(m)—NR₇R₈, O—(CH₂)_(n)—NR₇R₈, NH—C(O)—R₇, O—(CH₂)_(n)—NH—C(O)—R₇,O—(CH₂)_(n)—C(O)—NH—R₇, (CH₂)_(m)—C(O)R₇ and (CH₂)_(m)—C(O)NR₇R₈.

Suitably R₁ is selected from R₇, OR₇, CO₂R₇, NR₇R₈, NH—C(O)—R₇,O—(CH₂)_(n)—NH—C(O)—R₇, O—(CH₂)_(n)—C(O)—NH—R₇, C(O)R₇ and C(O)NR₇R₈.

Suitably R₁ is selected from R₇, OR₇, CO₂R₇, O—(CH₂)_(n)—NH—C(O)—R₇,O—(CH₂)_(n)—C(O)—NH—R₇, C(O)R₇ and C(O)NR₇R₈.

Suitably R₁ is selected from R₇, O—(CH₂)_(n)—NH—C(O)—R₇ andO—(CH₂)_(n)—C(O)—NH—R₇.

In some embodiments R₁ is H.

R₂

R₂ is selected from R₉, (CH₂)_(r)—OR₉, (CH₂)_(r)—CO₂R₉,(CH₂)_(r)—NR₉R₁₀, O—(CH₂)_(s)—NR₉R₁₀, NH—C(O)—R₉,O—(CH₂)_(s)—NH—C(O)—R₉, O—(CH₂)_(s)—C(O)—NH—R₉, (CH₂)_(r)—SO₂R₉,O—SO₂R₉, (CH₂)_(r)—COR₉ and (CH₂)_(r)—C(O)NR₉R₁₀.

Suitably R₂ is selected from R₉, (CH₂)_(r)—OR₉, (CH₂)_(r)—CO₂R₉,(CH₂)_(r)—NR₉R₁₀, O—(CH₂)_(s)—NR₉R₁₀, NH—C(O)—R₉,O—(CH₂)_(s)—NH—C(O)—R₉, O—(CH₂)_(s)—C(O)—NH—R₉, (CH₂)_(r)—COR₉ and(CH₂)_(r)—C(O)NR₉R₁₀.

Suitably R₂ is selected from R₉, OR₉, CO₂R₉, NR₉R₁₀, NH—C(O)—R₉,O—(CH₂)_(s)—NH—C(O)—R₉, O—(CH₂)_(s)—C(O)—NH—R₉, COR₉ and C(O)NR₉R₁₀.

Suitably R₂ is selected from R₉, OR₉, CO₂R₉, O—(CH₂)_(s)—NH—C(O)—R₉,O—(CH₂)_(s)—C(O)—NH—R₉, COR₉ and C(O)NR₉R₁₀.

Suitably R₂ is selected from R₉, O—(CH₂)_(s)—NH—C(O)—R₉ andO—(CH₂)_(s)—C(O)—NH—R₉.

In some embodiments R₂ is H.

R₃

Suitably R₃ is selected from H, C₁₋₆ alkyl and CH₂Ph.

Suitably R₃ is selected from H, methyl, ethyl and CH₂Ph.

More suitably R₃ is selected from methyl and ethyl.

More suitably R₃ is methyl.

R₄

R₄ is selected from phenyl and C₅₋₉ heteroaryl groups optionallysubstituted with up to three optional substituent groups. Hence, any ofthe phenyl group or the C₅₋₉ heteroaryl groups selected for R₄ may beoptionally substituted with up to three optional substituent groups.

Suitably R₄ is selected from phenyl, pyrrolyl, N—methylpyrrolyl,furanyl, thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl,thiazolyl, pyridyl, benzofuranyl, benzothiophenyl, benzimidazolyl,N—methylbenzoimidazolyl, benzooxazolyl and benzothiazolyl, optionallysubstituted with up to three optional substituent groups selected fromOH, C₁₋₆ alkyl, OC₁₋₆ alkyl, (CH₂)_(j)—CO₂R₁₁, O—(CH₂)_(k)—NR₁₁R₁₂,(CH₂)_(j)—NR₁₁R₁₂, C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂; C(═O)—NH—R₂₄ andC(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂.

Suitably R₄ is selected from phenyl, pyrrolyl, N—methylpyrrolyl,furanyl, thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl,thiazolyl, benzofuranyl, benzothiophenyl, benzimidazolyl,N—methylbenzoimidazolyl, benzooxazolyl and benzothiazolyl, optionallysubstituted with one or two optional substituent groups selected fromOH, C₁₋₆ alkyl, OC₁₋₆ alkyl, (CH₂)_(j)—CO₂R₁₁, O—(CH₂)_(k)—NR₁₁R₁₂,(CH₂)_(j)—NR₁₁R₁₂, C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂; C(═O)—NH—R₂₄ andC(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂.

Suitably R₄ is selected from phenyl, N—methylpyrrolyl, thiophenyl,N—methylimidazolyl, oxazolyl, thiazolyl, benzothiophenyl,N—methylbenzoimidazolyl and benzothiazolyl, optionally substituted withone or two optional substituent groups selected from OH, C₁₋₆ alkyl,OC₁₋₆ alkyl, (CH₂)_(j)—CO₂R₁₁O—(CH₂)_(k)—NR₁₁R₁₂, (CH₂)_(j)—NR₁₁R₁₂,C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂; C(═O)—NH—R₂₄ andC(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂.

Suitably R₄ is optionally substituted with up to three optionalsubstituent groups selected from OH, C₁₋₆ alkyl, OC₁₋₆ alkyl,(CH₂)—CO₂R₁₁, O—(CH₂)_(k)—NH₂, (CH₂)_(j)—NH₂, C(═O)—NH—(CH₂)_(k)—NH₂;C(═O)—NH—R₂₄ and C(═O)—NH—(CH₂)_(k)—C(═NH)NH₂.

Suitably R₄ is an optionally substituted C(═O)—NH—R₂₄, wherein R₂₄ is—C₆H₄—(CH₂)_(j)—R₁₈, and the phenylene group —C₆H₄— is para substituted.

Suitably R₄ is optionally substituted with up to three optionalsubstituent groups selected from OH, methyl, ethyl, OCH₃, OCH₂CH₃, CO₂H,CO₂CH₃, CO₂CH₂CH₃, O—(CH₂)_(k)—NH₂ and (CH₂)_(j)—NH₂.

Suitably R₄ is optionally substituted with one or two optionalsubstituent groups.

More suitably R₄ is optionally substituted with one optional substituentgroup.

More suitably R₄ is selected from:

wherein Z₁ is selected from NH, N—CH₃, S and O;

Z₂ is selected from CH and N;

Z₃ is selected from S and O;

Z₄ is selected from CH and N;

R₂₂ is selected from (CH₂)_(j)CO₂R₁₁, (CH₂)_(j)NR₁₁R₁₂ andC(═O)—NH—C₆H₄—(CH₂)_(j)—R₁₈;

R₁₈ is selected from H, CO₂R₁₁ and NR₁₁R₁₂;

j is selected from an integer from 0 to 6;

R₁₁ and R₁₂ are independently selected from H and C₁₋₆ alkyl; and

R₂₃ is selected from H and C₁₋₆ alkyl.

The wavy line indicates the point of attachment of the above R₄ group tothe rest of the compound of formula (I).

More suitably R₄ is selected from:

wherein Z₁ is selected from NH, N—CH₃, S and O;

Z₂ is selected from CH and N; and

Z₃ is selected from S and O;

Z₄ is selected from CH and N;

R₁₁ is selected from H and C₁₋₆ alkyl; and

R₂₃ is selected from H and C₁₋₆ alkyl.

R₅ and R₆

Suitably for (iii) R₅ is H and R₆ is an OC₁₋₆ alkyl selected from O—CH₃and O—CH₂CH₃.

Most suitably, (i) R₅ and R₆ together form a double bond.

R₇

Suitably R₇ is selected from H, C₁₋₁₂ alkyl, C₅₋₉ heteroaryl, C₆₋₁₅heteroarylalkyl, phenyl, benzyl and phenethyl; wherein the heteroaryl,heteroarylalkyl, phenyl and aralkyl groups are optionally substitutedwith up to three groups selected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

Suitably R₇ is selected from H, C₁₋₁₂ alkyl, pyrrolyl, N—methylpyrrolyl,furanyl, thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl,thiazolyl, pyridyl, indolyl, N—methylindolyl, benzofuranyl,benzothiophenyl, benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyl,benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl,imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3-ylmethyl,furan-3-ylmethyl, phenyl, benzyl and phenethyl; wherein the heteroaryl,heteroarylalkyl, phenyl and aralkyl groups are optionally substitutedwith up to three groups selected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

Suitably R₇ is selected from H, C₁₋₆ alkyl, pyrrolyl, N—methylpyrrolyl,furanyl, thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl,thiazolyl, pyridyl, indolyl, N—methylindolyl, benzofuranyl,benzothiophenyl, benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyl,benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl,imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3-ylmethyl,furan-3-ylmethyl, phenyl, benzyl and phenethyl; wherein the heteroaryl,heteroarylalkyl, phenyl and aralkyl groups are optionally substitutedwith up to three groups selected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

Suitably R₇ is selected from H, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, i-butyl, t-butyl, pyrrolyl, N—methylpyrrolyl, furanyl,thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl, thiazolyl,pyridyl, indolyl, N—methylindolyl, benzofuranyl, benzothiophenyl,benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl,pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, imidazol-2-ylmethyl,imidazol-4-ylmethyl, thiophen-3-ylmethyl, furan-3-ylmethyl, phenyl,benzyl and phenethyl optionally substituted with up to three groupsselected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

Suitably R₇ is selected from H, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, i-butyl, t-butyl, pyrrolyl, N—methylpyrrolyl, furanyl,thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl, thiazolyl,pyridyl, indolyl, N—methylindolyl, benzofuranyl, benzothiophenyl,benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl,phenyl, benzyl and phenethyl optionally substituted with up to threegroups selected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

In some embodiments, R₇ is selected from H, methyl, ethyl, n-propyl,i-propyl, n-butyl, s-butyl, i-butyl, t-butyl.

R₉

Suitably R₉ is selected from H, C₁₋₁₂ alkyl, C₅₋₉ heteroaryl, C₆₋₁₅heteroarylalkyl, phenyl, benzyl and phenethyl; wherein the heteroaryl,heteroarylalkyl, phenyl and aralkyl groups are optionally substitutedwith up to three groups selected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

Suitably R₉ is selected from H, C₁₋₁₂ alkyl, pyrrolyl, N—methylpyrrolyl,furanyl, thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl,thiazolyl, pyridyl, indolyl, N—methylindolyl, benzofuranyl,benzothiophenyl, benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyl,benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl,imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3-ylmethyl,furan-3-ylmethyl, phenyl, benzyl and phenethyl; wherein the heteroaryl,heteroarylalkyl, phenyl and aralkyl groups are optionally substitutedwith up to three groups selected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

Suitably R₉ is selected from H, C₁₋₆ alkyl, pyrrolyl, N—methylpyrrolyl,furanyl, thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl,thiazolyl, pyridyl, indolyl, N—methylindolyl, benzofuranyl,benzothiophenyl, benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyl,benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl,imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3-ylmethyl,furan-3-ylmethyl, phenyl, benzyl and phenethyl; wherein the heteroaryl,heteroarylalkyl, phenyl and aralkyl groups are optionally substitutedwith up to three groups selected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

Suitably R₉ is selected from H, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, i-butyl, t-butyl, pyrrolyl, N—methylpyrrolyl, furanyl,thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl, thiazolyl,pyridyl, indolyl, N—methylindolyl, benzofuranyl, benzothiophenyl,benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl,pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, imidazol-2-ylmethyl,imidazol-4-ylmethyl, thiophen-3-ylmethyl, furan-3-ylmethyl, phenyl,benzyl and phenethyl optionally substituted with up to three groupsselected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

Suitably R₉ is selected from H, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, i-butyl, t-butyl, pyrrolyl, N—methylpyrrolyl, furanyl,thiophenyl, imidazolyl, N—methylimidazolyl, oxazolyl, thiazolyl,pyridyl, indolyl, N—methylindolyl, benzofuranyl, benzothiophenyl,benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl,phenyl, benzyl and phenethyl optionally substituted with up to threegroups selected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl.

In some embodiments, R₉ is selected from H, methyl, ethyl, n-propyl,i-propyl, n-butyl, s-butyl, i-butyl, t-butyl.

R₈, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₂₀ and R₂₁

Suitably each of R₈, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₂₀ and R₂₁are independently selected from H, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, i-butyl and t-butyl.

Suitably each of R₈, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₂₀ and R₂₁are independently selected from H, methyl, and ethyl.

Suitably R₈ is H.

Suitably R₁₀ is H.

Suitably each R₁₁ is independently selected from H and methyl.

Suitably each R₁₂ is independently selected from H and methyl; moresuitably each R₁₂ is H.

Suitably R₁₃ is H.

Suitably R₁₄ is H.

Suitably R₁₅ is H.

Suitably R₁₆ is H.

Suitably R₁₇ is methyl.

Suitably R₂₀ is H.

Suitably R₂₁ is H.

R₁₈

Suitably R₁₈ is selected from H, CO₂H, CO₂CH₃, CO₂CH₂CH₃, NH(CH₃) andNH₂.

R₁₉

Suitably R₁₉ is selected from H, (CH₂)_(t)—N(CH₂CH₃)₂,(CH₂)_(t)—N(CH₃)₂, (CH₂)_(t)—NH(CH₂CH₃), (CH₂)_(t)—NH(CH₃) and(CH₂)_(t)—NH₂.

More suitably R₁₉ is selected from H and (CH₂)_(t)—NH₂.

R₂₄

Suitably, R₂₄ is a phenyl optionally substituted with up to threeoptional substituent groups selected from OH, methyl, ethyl, propyl,OCH₃, OCH₂CH₃, CO₂H, CO₂CH₃, CO₂CH₂CH₃, O—(CH₂)_(k)—NH₂,O—(CH₂)_(k)—NH(CH₃), (CH₂)_(j)—NH₂, (CH₂)_(j)—NH(CH₃),C(═O)—NH—(CH₂)_(k)—NH₂, C(═O)—NH—(CH₂)_(k)—NH(CH₃),C(═O)—NH—(CH₂)_(k)—C(═NH)NH(CH₃), and C(═O)—NH—(CH₂)_(k)—C(═NH)NH₂.

Suitably, R₂₄ is a phenyl optionally substituted with up to threeoptional substituent groups selected from OH, methyl, ethyl, OCH₃,OCH₂CH₃, CO₂H, CO₂CH₃, CO₂CH₂CH₃, O—(CH₂)_(k)—NH₂ and (CH₂)_(j)—NH₂.

Suitably, R₂₄ is a para substituted phenyl group.

More suitably, in some aspects R₂₄ is —C₆H₄—(CH₂)_(j)—R₁₈, wherein R₁₈is selected from CO₂R₁₁ and NR₁₁R₁₂.

j

Each instance of j is independently selected from an integer from 0 to6, hence, each j is independently selected from 0, 1, 2, 3, 4, 5 and 6.

Suitably each j is independently selected from 0, 1, 2 and 3.

More suitably each j is independently selected from 0 and 1.

More suitably each j is 0.

k

Each instance of k is independently selected from an integer from 1 to6, hence, each k is independently selected from 1, 2, 3, 4, 5 and 6.

Suitably each k is independently selected from 1, 2 and 3.

More suitably each k is 1.

m

m is selected from an integer from 0 to 6, hence, m is selected from 0,1, 2, 3, 4, 5 and 6.

Suitably m is selected from 0, 1, 2 and 3.

More suitably m is selected from 0 and 1.

More suitably m is 0.

n

n is selected from an integer from 1 to 6, hence, n is selected from 1,2, 3, 4, 5 and 6.

Suitably n is selected from 1, 2 and 3.

More suitably n is 1.

r

r is selected from an integer from 0 to 6, hence, r is selected from 0,1, 2, 3, 4, 5 and 6.

Suitably r is selected from 0, 1, 2 and 3.

More suitably r is selected from 0 and 1.

More suitably r is 0.

s

s is selected from an integer from 1 to 6, hence, s is selected from 1,2, 3, 4, 5 and 6.

Suitably s is selected from 1, 2 and 3.

More suitably s is 1.

t

t is selected from an integer from 0 to 6, hence, t is selected from 0,1, 2, 3, 4, 5 and 6.

Suitably t is selected from 0, 1, 2 and 3.

More suitably t is selected from 0 and 1.

More suitably t is 0.

Y₁

Y₁ is N or CH; suitably Y₁ is CH.

Y₂

Y₂ is N or CH; suitably Y₂ is CH.

X₁

Suitably X₁ is selected from O, S, NH, CH₂, CH₂O, C(═O), C(═O)NR₁₃,NR₁₃C(═O), O—C(O) and C(O)—O;

Suitably, X₁ is selected from O, C(═O), C(═O)NR₁₃ and NR₁₃C(═O).

More suitably X₁ is selected from O, C(═O)NH and NHC(═O).

More suitably X₁ is O.

X₂

Suitably X₂ is selected from O, S, NH, CH₂, CH₂O, C(═O), C(═O)NR₁₅,NR₁₅C(═O), O—C(O) and C(O)—O or is absent.

Suitably X₂ is selected from O, C(═O), C(═O)NR₁₅ and NR₁₆C(═O) or isabsent.

More suitably X₂ is selected from O, C(═O)NH and NHC(═O).

Suitably X₂ is the same as X₁.

More suitably X₂ is O.

L

L is a linker group. Suitably, any of the peptide chain, alkylene chain,paraformaldehyde chain or polyethylene glycol chain is interrupted byone or more hetero-atoms (e.g., N, O and S) and/or one or more C₅₋₉heteroarylene groups (e.g., pyrrolylene, pyrazolylene, pyrazolylene,1,2,3-triazolylene, pyridinylene) and/or one or more phenylene group.More suitably, the chains may be interrupted by from one to threehetero-atoms and/or from one to three C₅₋₉ heteroarylene groups and/orfrom one to three phenylene groups. Suitably, when L is any one of thechains described above, p is 1, q is 1, 2, 3, 4, 5, or 6, or p is 1 andq is, 2, 3, 4, 5, or 6.

Suitably L is selected from a peptide chain having from 2 to 5 aminoacids, from 2 to 4 amino acids, from 2 to 3 amino acids; an alkylenechain containing from 1 to 11 carbon atoms, from 1 to 10 carbon atoms,from 1 to 9 carbon atoms, from 1 to 8 carbon atoms, from 1 to 7 carbonatoms, from 1 to 6 carbon atoms, from 1 to 5 carbon atoms, from 1 to 4carbon atoms, from 1 to 3 carbon atoms, which may contain one or morecarbon-carbon double or triple bonds; a paraformaldehyde chain—(OCH₂)₁₋₁₂—, —(OCH₂)₁₋₁₁—, —(OCH₂)₁₋₁₀—, —(OCH₂)₁₋₉—, —(OCH₂)₁₋₈—,—(OCH₂)₁₋₇—, —(OCH₂)₁₋₆—, —(OCH₂)₁₋₅—, —(OCH₂)₁₋₄—, —(OCH₂)₁₋₃— apolyethylene glycol chain —(OCH₂CH₂)₁₋₅—, chain —(OCH₂CH₂)₁₋₄—, chain—(OCH₂CH₂)₁₋₃—; which chain may be interrupted by one or morehetero-atoms and/or C₅₋₉ heteroarylene groups and/or from one to threephenylene groups. Suitably, when L is any one of the chains describedabove, p is 1, q is 1, 2, 3, 4, 5, or 6, or p is 1 and q is, 2, 3, 4, 5,or 6.

More suitably, L may be selected from an alkylene chain containing from1 to 12 carbon atoms which may contain one or more carbon-carbon doubleor triple bonds.

More suitably, L may be selected from CH═CH, CH₂, CH₂CH₂, CH₂CH₂CH₂,CH₂CH₂CH₂CH₂ and CH₂CH₂CH₂CH₂CH₂.

A

In one embodiment A is A1:

wherein for each A1 group one of Y₃ and Y₄ is independently selectedfrom N—R₁₇, S and O; and the other of Y₃ and Y₄ is CH; and Y₅ isindependently selected from CH, N, S and COH.

In this embodiment, when q is selected from 2, 3, 4, 5 and 6 then A willcontain multiple A1 groups connected to each other.

Hence, the 5-membered ring containing Y₃, Y₄ and Y₅ is a heteroarylring. This A1 group may be attached to the rest of the molecule ineither direction. Hence, when A is A1, as in the above embodiment, thecompound of formula (I) is selected from:

More suitably, when A is A1 the compound of formula (I) is compound(II).

Hence, the heteroaryl ring containing Y₃, Y₄ and Y₅, is selected fromone of the following groups:

More suitably A is

wherein Y₅ is selected from CH and N.

In another embodiment A is A2:

wherein for each A2 group one of Y₆ and Y₇ is independently selectedfrom N and CH; and the other of Y₆ and Y₇ is CH.

Hence, the 6-membered ring containing Y₆ and Y₇ is a phenyl or pyridinylring. The A2 group may be attached to the rest of the molecule in eitherdirection. Hence, when A is A2, as in the above embodiment, the compoundof formula (I) is selected from:

More suitably, when A is A2 the compound of formula (I) is compound(IV).

Suitably, A is A4:

More suitably Y₆ is CH; and Y₇ is CH.

q

Suitably q is selected from 0, 1, 2 and 3.

More suitably q is 0 or 1.

6-Membered Aromatic-Ring

Suitably, the 6-membered aromatic ring of formula (I) ispara-substituted:

More suitably, the 6-membered aromatic ring of formula (I) is:

Optional Double Bonds in the C-Ring

The present invention provides a compound of formula (I):

wherein the dotted lines indicates the optional presence of a doublebond between one or more of C1 and C2, C2 and C3, and C3 and C4.

In one aspect, the compound of formula (I) has a double bond between C1and C2 to give a compound of formula (VI):

In another aspect, the compound of formula (I) has a double bond betweenC2 and C3 to give a compound of formula (VII):

In another aspect, the compound of formula (I) has a double bond betweenC3 and C4 to give a compound of formula (VIII):

In another aspect, the compound of formula (I) has a double bond betweenC1 and C2 and a double bond between C3 and C4 to give a compound offormula (IX):

Other Limitations

The options for compounds of formula (I) contain the proviso that when pis 0 and A is A1, then: (a) for at least one A1 group one of Y₃ and Y₄is selected from S and O; or (b) for at least one A1 group Y₅ is S; or(c) R₄ is not an optionally substituted pyrrolyl or imidazolyl.

There will be more than one A1 group when q is selected from 2, 3, 4, 5and 6.

Hence, when p is 0 and A is A1, the proviso requires the presence of atleast one aryl group or, alternatively, the presence of a heteroarylgroup (either as part of A1 or R₄) which does not contain a 5-memberedpyrrole or imidazole ring, or optionally substituted derivatives such asN—methylpyrrole or N—methylimidazole rings. As a result, this provisoprevents the compounds of formula (I) having a purely poly-pyrrole orpoly-imidazole or poly-pyrrole-imidazole long chain group attached tothe PDD. Compounds having such long chain groups tend to be relativelypoorly cytotoxic.

In some aspects, suitably the options for compounds of formula (I)contains the proviso that when p is 0 and A is A1, then: (a) the5-membered ring of A1 is selected from H9, H10, H11, H12, H13, H14, H15,H16, H17, H19, H20, H21, H22, H23 and H24; or (b) the 5-membered ring ofA1 is selected from H5 and H6; or (c) R₄ is selected from phenyl,furanyl, thiophenyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl,benzothiophenyl, benzimidazolyl, N—methylbenzoimidazolyl, benzooxazolyland benzothiazolyl, optionally substituted with up to three optionalsubstituent groups selected from OH, C₁₋₆ alkyl, OC₁₋₆ alkyl,(CH₂)_(j)—CO₂R₁₁, O—(CH₂)_(k)—NR₁₁R₁₂, (CH₂)_(j)—NR₁₁R₁₂,C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂; C(═O)—NH—C₆H₄—(CH₂)_(j)—R₁₈ andC(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂.

In some aspects, suitably the options for compounds of formula (I)contains the proviso that when p is 0 and A is A1, then: (a) the5-membered ring of A1 is selected from H9, H10, H11, H12, H13, H14, H15,H16, H17, H19, H20, H21, H22, H23 and H24; or (b) the 5-membered ring ofA1 is selected from H5 and H6; or (c) R₄ is selected from phenyl and C₉heteroaryl groups optionally substituted with up to three optionalsubstituent groups selected from OH, C₁₋₆ alkyl, OC₁₋₆ alkyl,(CH₂)_(j)—CO₂R₁₁, O—(CH₂)_(k)—NR₁₁R₁₂, (CH₂)_(j)—NR₁₁R₁₂,C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂; C(═O)—NH—C₆H₄—(CH₂)_(j)—R₁₈;C(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂, with the proviso that the C₅₋₉heteroaryl is not indolyl.

In some aspects, suitably the options for compounds of formula (I)contains the proviso that i) L is a peptide chain having from 2 to 6amino acids, an alkylene chain containing from 1 to 12 carbon atomswhich may contain one or more carbon-carbon double or triple bonds, aparaformaldehyde chain, a polyethylene glycol chain, which chains areinterrupted by one or more of C₅₋₉ heteroarylene, phenylene orcombinations of the foregoing; and/or ii) p is 1; and/or iii) q is 1, 2,3, 4, 5 or 6.

Suitable Structures

The compound of formula (I):

is drawn without specifying the position of R₁ and R₂ on the C-ring.Hence, R₁ and R₂ may be present on any position of the C-ring providedthat the valence requirement are met. As the fused carbon and thenitrogen of the C-ring have all their substituents shown, this meansthat R₁ and R₂ may be present on any of the non-fused carbons of theC-ring (i.e. the C1, C2, C3 or C4 positions as designated above).Suitably R₁ and R₂ are present on two different non-fused carbons of theC-ring.

In one aspect, the compound of formula (I) is selected from:

In another aspect, the compound of formula (I) is selected from:

More suitably, the compound of formula (I) has the following structure:

For compounds of formula (XV) where R₁ and/or R₂ are substituents otherthan H, the carbons in the C-ring to which any such substituents areattached will be stereocenters. In formula (XV) R₁ and R₂ are drawnwithout specifying the stereochemistry of the carbons on the C-ring towhich they are attached.

More suitably the compound of formula (I) is selected from:

wherein q is selected from 0, 1, 2, 3, 4, 5 or 6;

p is 0 or 1;

L is an alkylene chain containing from 1 to 12 carbon atoms;

Y₁ is N or CH;

Y₂ is N or CH; and wherein at least one of Y₁ and Y₂ is CH;

Y₅ is selected from CH and N;

Z₁ is selected from O, S, NH and N—CH₃;

Z₂ is selected from CH and N;

Z₃ is selected from S and O;

Z₄ is selected from CH and N;

R₂₂ is selected from (CH₂)_(j)CO₂H, (CH₂)_(j)CO₂C₁₋₆ alkyl,(CH₂)_(j)NR₁₁R₁₂ and C(═O)—NH—C₆H₄—(CH₂)_(j)—R₁₈;

R₁₈ is selected from H, CO₂R₁₁ and NR₁₁R₁₂;

R₁₉ is selected from H and (CH₂)_(t)—NR₂₀R₂₁;

j and t are independently selected from an integer from 0 to 6; and

R₁₁, R₁₂ and R₂₃ are independently selected from H and C₁₋₆ alkyl.

and either:

-   -   (i) R₅ and R₆ together form a double bond;    -   (ii) R₅ is H and R₆ is OH; or    -   (iii) R₅ is H and R₆ is OC₁₋₆ alkyl;

with the proviso that when the compound is (XVI) and p is 0, that Z₁ isselected from O and S.

More suitably the compound of formula (I) is selected from:

wherein q is selected from 0, 1, 2, 3, 4, 5 or 6;

-   -   p is 0 or 1;

L is an alkylene chain containing from 1 to 12 carbon atoms;

Y₁ is N or CH;

Y₂ is N or CH; and wherein at least one of Y₁ and Y₂ is CH;

Y₅ is selected from CH and N;

Z₁ is selected from O, S, NH and N—CH₃;

Z₂ is selected from CH and N;

Z₃ is selected from S and O;

Z₄ is selected from CH and N;

R₁₉ is selected from H and (CH₂)_(t)—NR₂₀R₂₁;

t is selected from an integer from 0 to 6;

R₁₁, R₂₀, R₂₁ and R₂₃ are independently selected from H and C₁₋₆ alkyl;

and either:

-   -   (i) R₅ and R₆ together form a double bond;    -   (ii) R₅ is H and R₆ is OH; or    -   (iii) R₅ is H and R₆ is OC₁₋₆ alkyl;

with the proviso that when the compound is (XX) and p is 0, that Z₁ isselected from O and S.

More suitably, the compound of formula (I) is selected from:

-   (a)    methyl(S)—5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)benzo-[b]thiophene-2-carboxylate    (13)

-   (b)    methyl(S)—5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)-benzo[b]thiophene-2-carboxylate    (17)

-   (c)    methyl(S)—4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate    (20)

-   (d)    methyl(S)—4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate    (24)

-   (e)    methyl(S)—4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-benzamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate    (28)

-   (f) methyl    (S)—5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-benzamido)benzo[b]thiophene-2-carboxylate    (30)

-   (g) methyl    (S)—4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)-benzoate    (34)

-   (h) methyl    (S)—4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)-benzoate    (38)

-   (i)    (S)—N—(4-aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butan-amido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide    (41)

-   (j)    (S)—N—(2-((4-Aminophenyl)carbamoyl)benzo[b]thiophen-5-yl)-4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)-butanamido)-1-methyl-1H-pyrrole-2-carboxamide    (47)

-   (S)—N—(4-aminophenyl)-4-(4-(4((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydroxybenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexan-amido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide    (62).

-   (l)    (S)—N—(4-Aminophenyl)-4-(6-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexan-amido)-1-methyl-1H-pyrrole-2-carboxamide    (66)

and

-   (m)    (S)—N—(2-Aminoethyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide    (68)

In a further aspect, there is provided a compound of formula (I):

and salts and solvates thereof, wherein:

the dotted lines indicates the optional presence of a double bondbetween one or more of C1 and C2, C2 and C3, and C3 and C4;

R₁ is selected from R₇, ═CH₂, ═CH—(CH₂)_(m)—CH₃, ═O, (CH₂)_(m)—OR₇,(CH₂)_(m)—CO₂R₇, (CH₂)_(m)—NR₇R₈, O—(CH₂)_(n)—NR₇R₈, NH—C(O)—R₇,O—(CH₂)_(n)—NH—C(O)—R₇, O—(CH₂)_(n)—C(O)—NH—R₇, (CH₂)_(m)—SO₂R₇,O—SO₂R₇, (CH₂)_(m)—C(O)R₇ and (CH₂)_(m)—C(O)NR₇R₈;

R₂ is selected from R₉, ═CH₂, ═CH—(CH₂)_(r)—CH₃, ═O, (CH₂)_(r)—OR₉,(CH₂)_(r)—CO₂R₉, (CH₂)_(r)—NR₉R₁₀, O—(CH₂)_(s)—NR₉R₁₀, NH—C(O)—R₉,O—(CH₂)_(s)—NH—C(O)—R₉, O—(CH₂)_(s)—C(O)—NH—R₉, (CH₂)_(r)—SO₂R₉,O—SO₂R₉, (CH₂)_(r)—COR₉ and (CH₂)_(r)—C(O)NR₉R₁₀;

R₃ is selected from H, C₁₋₁₂ alkyl and CH₂Ph;

R₄ is selected from phenyl and C₅₋₉ heteroaryl groups optionallysubstituted with up to three optional substituent groups selected fromOH, C₁₋₆ alkyl, OC₁₋₆ alkyl, (CH₂)_(j)—CO₂R₁₁, O—(CH₂)_(k)—NR₁₁R₁₂,(CH₂)_(j)—NR₁₁R₁₂, C(═O)—NH—(CH₂)_(k)—NR₁₁R₁₂;C(═O)—NH—C₆H₄—(CH₂)_(j)—R₁₈ and C(═O)—NH—(CH₂)_(k)—C(═NH)NR₁₁R₁₂;

R₁₉ is selected from H and (CH₂)_(t)—NR₂₀R₂₁;

Y₁ is N or CH; Y₂ is N or CH; and wherein at least one of Y₁ and Y₂ isCH;

p is 0 or 1; j, m, r and t are independently selected from an integerfrom 0 to 6;

k, n and s are independently selected from an integer from 1 to 6;

X₁ is selected from O, S, NR₁₃, CR₁₃R₁₄, CR₁₃R₁₄O, C(═O), C(═O)NR₁₃,NR₁₃C(═O), O—C(O) and C(O)—O;

L is selected from an amino acid, a peptide chain having from 2 to 6amino acids, an alkylene chain containing from 1 to 12 carbon atomswhich may contain one or more carbon-carbon double or triple bonds, aparaformaldehyde chain —(OCH₂)₁₋₁₂—, a polyethylene glycol chain—(OCH₂CH₂)₁₋₆—, which chains may be interrupted by one or more of O, Sand/or NH groups and/or C₃₋₉ heteroarylene and/or phenylene;

X₂ is selected from O, S, NR₁₅, CR₁₅R₁₆, CR₁₅R₁₆O, C(═O), C(═O)NR₁₅,NR₁₅C(═O), O—C(O) and C(O)—O or is absent;

q is selected from 0, 1, 2, 3, 4, 5 and 6;

A is selected from:

-   -   for each A1 group one of Y₃ and Y₄ is independently selected        from N—R₁₇, S and O; and the other of Y₃ and Y₄ is CH; and Y₅ is        independently selected from CH, N, S and COH; and    -   for each A2 group one of Y₆ and Y₇ is independently selected        from N and CH; and the other of Y₆ and Y₇ is CH;

R₇ and R₉ are independently selected from H, C₁₋₁₂ alkyl, C₅₋₉heteroaryl, C₆₋₁₅ heteroarylalkyl, phenyl and C₇₋₁₂ aralkyl groups;wherein the heteroaryl, heteroarylalkyl, phenyl and aralkyl groups areoptionally substituted with up to three optional substituent groupsselected from C₁₋₆ alkyl, OH, OC₁₋₆ alkyl;

R₁₈ is selected from H, CO₂R₁₁ and NR₁₁R₁₂;

R₈, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₂₀ and R₂₁ areindependently selected from H and C₁₋₆ alkyl; and

(i) R₅ and R₆ together form a double bond; or (ii) R₅ is H and R₆ is OH;or (iii) R₅ is H and R₆ is OC₁₋₆ alkyl.

Applications

The invention finds application in the treatment of proliferativediseases.

In certain aspects a method of treating a proliferative disease isprovided, the method comprising administering to a subject atherapeutically effective amount of a compound of the formula (I) andsalts and solvates thereof or a composition comprising a compound offormula (I) and salts and solvates thereof.

In certain aspects a method of treating a proliferative disease isprovided, the method comprising administering to a subject atherapeutically effective amount of a targeted conjugate comprising acompound of the formula (I) and salts and solvates thereof.

In certain aspects a method of treating a proliferative disease isprovided, the method comprising administering to a subject atherapeutically effective amount of an antibody-drug conjugatecomprising a compound of the formula (I) and salts and solvates thereof.

The term “proliferative disease” refers to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo. Examples of proliferative conditions include, but are notlimited to, benign, pre-malignant, and malignant cellular proliferation,including but not limited to, neoplasms and tumours (e.g. histocytoma,glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lungcancer, hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, bowel cancer, colon cancer, hepatoma, breastcancer, glioblastoma, cervical cancer, ovarian cancer, oesophageal [oresophageal] cancer, oral cancer, prostate cancer, testicular cancer,liver cancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, uterine cancer, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, head and neck cancer,bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma,Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases,fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Suitably the proliferative disease is selected frombladder cancer, bone cancer, bowel cancer, brain cancer, breast cancer,cervical cancer, colon cancer, head and neck cancer, leukemia, livercancer, lung cancer, lymphoma, melanoma, oesophageal cancer, oralcancer, ovarian cancer, pancreatic cancer, prostate cancer, rectalcancer, renal cancer, retinoblastoma, sarcoma, skin cancer, stomachcancer, testicular cancer, thyroid cancer and uterine cancer. Suitablythe proliferative disease is selected from breast cancer and cervicalcancer.

Any type of cell may be treated, including but not limited to, bone,eye, head and neck, lung, gastrointestinal (including, e.g. mouth,oesophagus, bowel, colon), breast (mammary), cervix, ovarian, uterus,prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, andskin.

A skilled person is readily able to determine whether or not a candidatecompound treats a proliferative condition for any particular cell type.

Suitably subjects are human, livestock animals and companion animals.

In a further aspect, the compound of formula (I) and salts and solvatesthereof, may be linked, either directly or indirectly, to a targetingagent (e.g., antibody, antibody fragment, hormone, etc.) to provide atargeted conjugate. The target conjugates of the present disclosure maycontain one or multiple compounds of formula (I) (or salts and solvatesthereof). A variety of target conjugates are known in the art and may beused with a compound of formula (I) and salts and solvates thereof. Forexample, in a particular aspect the target conjugate is an antibody-drugconjugate, wherein one or more compounds of formula (I) are linked,directly or indirectly, to the antibody. Therefore, the compound offormula (I) and salts and solvates thereof, may be used as a payload ona targeted conjugate.

Suitably, a compound of formula (I) and salts and solvates thereof, foruse as a drug in targeted conjugate is prepared by attaching a compoundof formula (I) and salts and solvates thereof to a targeting agent,either directly or via an optional linker group.

Suitably, the compound of formula (I) and salts and solvates thereof, isattached to a targeting agent via a linker group. Suitably, the targetedconjugate is for use in the treatment of a disease, more specifically ofa proliferative disease. Suitably, the drug may be attached by anysuitable functional group that it contains to the targeting agent eitherdirectly or via a linker group. Typically, the drug contains, or can bemodified to contain, one or more functional groups such as amine,hydroxyl or carboxylic acid groups for attaching the drug to thetargeting agent either directly or via a linker group. In some aspects,one or more atoms or groups of the compound of formula (I) may beeliminated during the attachment of the drug to the antibody. In someaspects, the targeting agent binds to a cell surface receptor or atumor-associated antigen. In some aspects, the targeting agent is anantibody. In some aspects, the targeting agent is a hormone. In someaspects, the targeting agent is a protein. In some aspects, thetargeting agent is a polypeptide. In some aspects, the targeting agentis a small molecule (for example, folic acid).

The compounds of formula (I) find application as payloads for antibodiesor antibody fragments. The compounds of formula (I) readily allowconjugation to antibodies or antibody fragments.

Antibody Drug Conjugates

Antibody therapy has been established for the targeted treatment ofpatients with cancer, immunological and angiogenic disorders (Carter, P.(2006) Nature Reviews Immunology 6:343-357). The use of antibody-drugconjugates (ADC), i.e. immunoconjugates, for the local delivery ofcytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumorcells in the treatment of cancer, targets delivery of the drug moiety totumors, and intracellular accumulation therein, whereas systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells (Xie et al (2006)Expert. Opin. Biol. Ther. 6(3):281-291; Kovtun ef a/ (2006) Cancer Res.66(6):3214-3121; Law et al (2006) Cancer Res. 66(4):2328-2337; Wu et al(2005) Nature Biotech. 23(9): 1137-1145; Lambert J. (2005) Current Opin.in Pharmacol. 5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents15(9): 1087-1103; Payne, G. (2003) Cancer Cell 3:207-212; Trail ef a/(2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos(1999) Anticancer Research 19:605-614).

Maximal efficacy with minimal toxicity is sought thereby. Efforts todesign and refine ADC have focused on the selectivity of monoclonalantibodies (mAbs) as well as drug mechanism of action, drug-linking,drug/antibody ratio (loading), and drug-releasing properties (Junutula,et al., 2008b Nature Biotech., 26(8):925-932; Doman et al., (2009) Blood114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485; WO2009/052249;McDonagh (2006) Protein Eng. Design & Sel. 19(7): 299-307; Doronina etal., (2006) Bioconj. Chem. 17:114-124; Erickson et al., (2006) CancerRes. 66(8): 1-8; et al., (2005) Clin. Cancer Res. 11:843-852; Jeffrey etal., (2005) J. Med. Chem. 48:1344-1358; Hamblett et al., (2004) Clin.Cancer Res. 10:7063-7070).

In some aspects, the present invention relates to a compound of formula(I) and salts and solvates thereof, for use as a drug in anantibody-drug conjugate. Suitably, a compound of formula (I) and saltsand solvates thereof, for use as a drug in an antibody-drug conjugate isprepared by attaching a compound of formula (I) and salts and solvatesthereof to an antibody, either directly or via an optional linker group.Suitably, the compound of formula (I) and salts and solvates thereof, isattached to an antibody via a linker group. Suitably, the antibody-drugconjugate is for use in for treatment of a disease, more specifically ofa proliferative disease. Suitably, the antibody-drug conjugate is foruse in for treatment of a disease, more specifically of a proliferativedisease. Suitably, the drug may be attached by any suitable functionalgroup that it contains to the antibody either directly or via a linkergroup. Typically, the drug contains, or can be modified to contain, oneor more functional groups such as amine, hydroxyl or carboxylic acidgroups for attaching the drug to the antibody either directly or via alinker group. In some aspects, the antibody of the antibody drugconjugate is an antibody fragment, such as, but not limited to a singlechain antibody. In some aspects, one or more atoms or groups of thecompound of formula (I) may be eliminated during the attachment of thedrug to the antibody. In some aspects, the antibody binds to a cellsurface receptor or a tumor-associated antigen.

In some aspects, the present invention relates to a compound of formula(I) and salts and solvates thereof, for use in preparing a drug in anantibody-drug conjugate. Suitably, a compound of formula (I) and saltsand solvates thereof, may be used directly to prepare an antibody-drugconjugate when a compound of formula (I) and salts and solvates thereof,contains one or more functional groups (such as amine, hydroxyl orcarboxylic acid groups) for attaching the drug to the antibody eitherdirectly or via a linker group. Suitably, a compound of formula (I) andsalts and solvates thereof, may be used in preparing an antibody-drugconjugate by being modified to contain one or more functional groups(such as amine, hydroxyl or carboxylic acid groups) for attaching thedrug to the antibody either directly or via a linker group. Suitably, acompound of formula (I) and salts and solvates thereof, may be used inpreparing an antibody-drug conjugate by being modified to contain one ormore linker groups, wherein the antibody is attached to the drughthrough the one or more linker groups. Therefore, the present disclosureprovides for a compounds of the formula (I) further comprising one ormore linker group. Suitably, a compound of the formula (I) may contain1, 2, or 3 linker groups. Suitably, a compound of the formula (I) maycontain 1 or 2 linker groups. Suitably, a compound of the formula (I)may contain 1 linker group. In some aspects, one or more atoms or groupsof the compound of formula (I) may be eliminated during the attachmentof the drug to the antibody or the attachment of the linker to the drugor the modification of the drug to contain one or more functional groups(such as amine, hydroxyl or carboxylic acid groups) for attaching thedrug to the antibody either directly or via a linker group.

A variety of suitable linker groups are known in the art and may be usedas described herein. For example, the maleimide methodology is routinelyused as a method to attach antibodies to drug compounds by providing alinker attached to the drug with a terminal maleimide group. Examples143, 144 and 148 provide compounds of the formula (I) attached to linkergroups containing terminal maleimide groups. In addition, methodologiesusing diaryclyclooctyne moeities (such as, but not limited to, DBCO,dibenzylcyclooctyne) are also alternatives used in the art.Diarylcyclooctynes react with azides to provide attachment via theformation of stable triazoles. Diarylcyclooctynes are thermostable withvery narrow and specific reactivity toward azides, resulting in almostquantitative yields of stable triazoles. Furthermore, the reaction doesnot require a cytotoxic Cu(I) catalyst (that is toxic to most organisms)and thus, prevents its use in many biological systems. Still further,alkoxyamine methodologies are also alternatives used in the art. Forsite-specific conjugation of the drug to an antibody, the antibodies maycomprise a “tag” (which may be proprietary) that will react with adairylcyclooctyne (for example, DBCO), an alkyloxyamine and/or maleimidegroup to attach the antibody to the drug. The tag in some instances maybe a mutated amino acid. Suitable linker groups incorporating thevarious groups described above are available in the art. FIGS. 34A and Bprovides examples of compounds of the formula (I) (exemplified bycompound 140 of Example 143) attached to linker group containing anexemplary terminal alkoxyamine groups (FIG. 34A) and an exemplaryterminal diarylcyclooctyne group, DBCO (FIG. 34B).

In some aspects, the present invention relates to the use of a compoundof formula (I) and salts and solvates thereof, as a drug in anantibody-drug conjugate. Suitably, the use of a compound of formula (I)and salts and solvates thereof, as a drug in an antibody-drug conjugateis accomplished by attaching a compound of formula (I) and salts andsolvates thereof to an antibody, either directly or via an optionallinker group. Suitably, the compound of formula (I) and salts andsolvates thereof, is attached to an antibody via a linker group.Suitably, the antibody-drug conjugate is for use in for treatment of adisease, more specifically of a proliferative disease. Suitably, thedrug may be attached by any suitable functional group that it containsto the antibody either directly or via a linker group. Typically, thedrug contains, or can be modified to contain, one or more functionalgroups such as amine, hydroxyl or carboxylic acid groups for attachingthe drug to the antibody either directly or via a linker group. In someaspects, the antibody of the antibody drug conjugate is an antibodyfragment, such as, but not limited to a single chain antibody. In someaspects, one or more atoms or groups of the compound of formula (I) maybe eliminated during the attachment of the drug to the antibody. In someaspects, the antibody binds to a cell surface receptor or atumor-associated antigen.

The substituent groups of the compounds of formula (I) may interact withDNA sequences and may be selected so as to target specific sequences. Inparticular, the following groups in compounds of formula (I):

may be selected to target specific sequences. Hence, when thesubstituent groups are tailored in this way, the compounds of formula(I) find application in targeted chemotherapy.

Antibody and Antibody Fragments

The term “antibody” specifically covers monoclonal antibodies,polyclonal antibodies, dimers, multimers, multispecific antibodies(e.g., bispecific antibodies), intact antibodies and antibody fragments,so long as they exhibit the desired biological activity, for example,the ability to bind a desired antigen on a target cell or tissue.Antibodies may be murine, human, humanized, chimeric, or derived fromother species. An antibody is a protein generated by the immune systemthat is capable of recognizing and binding to a specific antigen.(Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology,5th Ed., Garland Publishing, New York). A target antigen generally hasnumerous binding sites, also called epitopes, recognized by CDRs on theantibody. Each antibody that specifically binds to a different epitopehas a different structure. Thus, one antigen may have more than onecorresponding antibody. An antibody includes a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin can be of anytype (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. lgG1, lgG2, lgG3,lgG4, lgA1 and lgA2) or subclass, or allotype (e.g. human G1m1, G1m2,G1m3, non-G1m1 [that, is any allotype other than G1 m1], G1m17, G2m23,G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6,G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2 and Km3) of immunoglobulinmolecule. The immunoglobulins can be derived from any species, includinghuman, murine, or rabbit origin.

As used herein, “binds an epitope” is used to mean the antibody binds anepitope with a higher affinity than a non-specific partner such asBovine Serum Albumin (BSA, Genbank accession no. CAA76847, version no.CAA76847.1 Gl:3336842, record update date: Jan. 7, 2011 02:30 PM). Insome embodiments the antibody binds an epitope with an associationconstant (Ka) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000,2000, 5000, 10⁴, 10⁵ or 10⁶-fold higher than the antibody's associationconstant for BSA, when measured at physiological conditions.

The term “antibody fragment” refers to a portion of a full lengthantibody, for example, the antigen binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and scFvfragments; diabodies; linear antibodies; fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies, CDR(complementary determining region), single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments andepitope-binding fragments of any of the above which immunospecificallybind to target antigens, such as, for example, cancer cell antigens,viral antigens or microbial antigens. The term “monoclonal antibody” asused herein refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e. the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site. Furthermore, in contrast to polyclonal antibodypreparations which include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant or epitope on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carryinga fully human immunoglobulin system (Lonberg (2008) Curr. Opinion20(4):450-459).

The antibodies, including monoclonal antibodies, herein specificallyinclude “chimeric” antibodies in which a portion of the antibodystructure, for example the heavy and/or light chain, is identical withor homologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984)Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies include“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g. Old World Monkey orApe) and human constant region sequences. An “intact antibody” herein isone comprising VL and VH domains, as well as a light chain constantdomain (CL) and heavy chain constant domains, CH₁, CH₂ and CH₃. Theconstant domains may be native sequence constant domains (e.g. humannative sequence constant domains) or amino acid sequence variantthereof. The intact antibody may have one or more “effector functions”which refer to those biological activities attributable to the Fc region(a native sequence Fc region or amino acid sequence variant Fc region)of an antibody. Examples of antibody effector functions include C1 qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; anddown regulation of cell surface receptors such as B cell receptor andBCR.

The antibodies disclosed herein may be modified. For example, to makethem less immunogenic to a human subject. This may be achieved using anyof a number of techniques familiar to the person skilled in the art,such as humanisation.

Administration & Dose

Compounds of formula I may be administered alone or in combination withone or another or with one or more pharmacologically active compoundswhich are different from the compounds of formula I.

Compounds of the invention may suitably be combined with variouscomponents to produce compositions of the invention. Suitably thecompositions are combined with a pharmaceutically acceptable carrier ordiluent to produce a pharmaceutical composition (which may be for humanor animal use). Suitable carriers and diluents include isotonic salinesolutions, for example phosphate-buffered saline. Useful pharmaceuticalcompositions and methods for their preparation may be found in standardpharmaceutical texts. See, for example, Handbook for PharmaceuticalAdditives, 3rd Edition (eds. M. Ash and I. Ash), 2007 (SynapseInformation Resources, Inc., Endicott, N.Y., USA) and Remington: TheScience and Practice of Pharmacy, 21st Edition (ed. D. B. Troy) 2006(Lippincott, Williams and Wilkins, Philadelphia, USA) which areincorporated herein by reference.

The compounds of the invention may be administered by any suitableroute. Suitably the compounds of the invention will normally beadministered orally or by any parenteral route, in the form ofpharmaceutical preparations comprising the active ingredient, optionallyin the form of a non-toxic organic, or inorganic, acid, or base,addition salt, in a pharmaceutically acceptable dosage form.

The compounds of the invention, their pharmaceutically acceptable salts,and pharmaceutically acceptable solvates of either entity can beadministered alone but will generally be administered in admixture witha suitable pharmaceutical excipient diluent or carrier selected withregard to the intended route of administration and standardpharmaceutical practice.

For example, the compounds of the invention or salts or solvates thereofcan be administered orally, buccally or sublingually in the form oftablets, capsules (including soft gel capsules), ovules, elixirs,solutions or suspensions, which may contain flavouring or colouringagents, for immediate-, delayed-, modified-, sustained-,controlled-release or pulsatile delivery applications. The compounds ofthe invention may also be administered via fast dispersing or fastdissolving dosages forms.

Such tablets may contain excipients such as microcrystalline cellulose,lactose, sodium citrate, calcium carbonate, dibasic calcium phosphateand glycine, disintegrants such as starch (preferably corn, potato ortapioca starch), sodium starch glycollate, croscarmellose sodium andcertain complex silicates, and granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, stearic acid, glycerylbehenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

Modified release and pulsatile release dosage forms may containexcipients such as those detailed for immediate release dosage formstogether with additional excipients that act as release rate modifiers,these being coated on and/or included in the body of the device. Releaserate modifiers include, but are not exclusively limited to,hydroxypropylmethyl cellulose, methyl cellulose, sodiumcarboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethyleneoxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer,hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetatephthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acidcopolymer and mixtures thereof. Modified release and pulsatile releasedosage forms may contain one or a combination of release rate modifyingexcipients. Release rate modifying excipients may be present both withinthe dosage form i.e. within the matrix, and/or on the dosage form i.e.upon the surface or coating.

Fast dispersing or dissolving dosage formulations (FDDFs) may containthe following ingredients: aspartame, acesulfame potassium, citric acid,croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate,ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesiumstearate, mannitol, methyl methacrylate, mint flavouring, polyethyleneglycol, fumed silica, silicon dioxide, sodium starch glycolate, sodiumstearyl fumarate, sorbitol, xylitol.

The compounds of the invention can also be administered parenterally,for example, intravenously, intra-arterially, or they may beadministered by infusion techniques. For such parenteral administrationthey are best used in the form of a sterile aqueous solution which maycontain other substances, for example, enough salts or glucose to makethe solution isotonic with blood. The aqueous solutions should besuitably buffered (preferably to a pH of from 3 to 9), if necessary. Thepreparation of suitable parenteral formulations under sterile conditionsis readily accomplished by standard pharmaceutical techniques well-knownto those skilled in the art.

Suitably formulation of the invention is optimised for the route ofadministration e.g. oral, intravenously, etc.

Administration may be in one dose, continuously or intermittently (e.g.in divided doses at appropriate intervals) during the course oftreatment. Methods of determining the most effective means and dosageare well known to a skilled person and will vary with the formulationused for therapy, the purpose of the therapy, the target cell(s) beingtreated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and the doseregimen being selected by the treating physician, veterinarian, orclinician.

Depending upon the disorder and patient to be treated, as well as theroute of administration, the compositions may be administered at varyingdoses. For example, a typical dosage for an adult human may be 100 ng to25 mg (suitably about 1 micro g to about 10 mg) per kg body weight ofthe subject per day.

Suitably guidance may be taken from studies in test animals whenestimating an initial dose for human subjects. For example when aparticular dose is identified for mice, suitably an initial test dosefor humans may be approx. 0.5× to 2× the mg/Kg value given to mice.

Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Isomers, Salts and Solvates

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; alpha- and beta-forms;axial and equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.

A reference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not apply to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol,amidine/amidine, nitroso/oxime, thioketone/enethiol,N—nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof.

Methods for the preparation (e.g. asymmetric synthesis) and separation(e.g. fractional crystallisation and chromatographic means) of suchisomeric forms are either known in the art or are readily obtained byadapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound alsoincludes ionic, salt, solvate, and protected forms of thereof, forexample, as discussed below.

In some embodiments, the compound of formula (I) and salts and solvatesthereof, comprises pharmaceutically acceptable salts of the compounds offormula (I).

Compounds of Formula (I), which include compounds specifically namedabove, may form pharmaceutically acceptable complexes, salts, solvatesand hydrates. These salts include nontoxic acid addition salts(including di-acids) and base salts.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then an acid addition salt may beformed with a suitable anion. Examples of suitable inorganic anionsinclude, but are not limited to, those derived from the followinginorganic acids hydrochloric acid, nitric acid, nitrous acid, phosphoricacid, sulfuric acid, sulphurous acid, hydrobromic acid, hydroiodic acid,hydrofluoric acid, phosphoric acid and phosphorous acids. Examples ofsuitable organic anions include, but are not limited to, those derivedfrom the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic,aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic,ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic,glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic,isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic,mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic,phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic,sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitablepolymeric organic anions include, but are not limited to, those derivedfrom the following polymeric acids: tannic acid, carboxymethylcellulose. Such salts include acetate, adipate, aspartate, benzoate,besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate,citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate,gluconate, glucuronate, hexafluorophosphate, hibenzate,hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,isethionate, lactate, malate, maleate, malonate, mesylate,methylsulfonate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate,oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogenphosphate, pyroglutamate, saccharate, stearate, succinate, tannate,tartrate, tosylate, trifluoroacetate and xinofoate salts.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO⁻), then a base salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, metal cations, such as an alkali or alkalineearth metal cation, ammonium and substituted ammonium cations, as wellas amines. Examples of suitable metal cations include sodium (Na⁺)potassium (K⁺), magnesium (Mg²⁺), calcium (Ca²⁺), zinc (Zn²⁺), andaluminum (Al³⁺). Examples of suitable organic cations include, but arenot limited to, ammonium ion (i.e. NH4⁺) and substituted ammonium ions(e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitablesubstituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺. Examples of suitable aminesinclude arginine, N,N′-dibenzylethylene-diamine, chloroprocaine,choline, diethylamine, diethanolamine, dicyclohexylamine,ethylenediamine, glycine, lysine, N—methylglucamine, olamine,2-amino-2-hydroxymethyl-propane-1,3-diol, and procaine. For a discussionof useful acid addition and base salts, see S. M. Berge et al., J.Pharm. Sci. (1977) 66:1-19; see also Stahl and Wermuth, Handbook ofPharmaceutical Salts: Properties, Selection, and Use (2011)

Pharmaceutically acceptable salts may be prepared using various methods.For example, one may react a compound of Formula 1 with an appropriateacid or base to give the desired salt. One may also react a precursor ofthe compound of Formula 1 with an acid or base to remove an acid- orbase-labile protecting group or to open a lactone or lactam group of theprecursor. Additionally, one may convert a salt of the compound ofFormula 1 to another salt through treatment with an appropriate acid orbase or through contact with an ion exchange resin. Following reaction,one may then isolate the salt by filtration if it precipitates fromsolution, or by evaporation to recover the salt. The degree ofionization of the salt may vary from completely ionized to almostnon-ionized.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate”describes a molecular complex comprising the compound and one or morepharmaceutically acceptable solvent molecules (e.g., EtOH). The term“hydrate” is a solvate in which the solvent is water. Pharmaceuticallyacceptable solvates include those in which the solvent may beisotopically substituted (e.g., D₂O, acetone-d6, DMSO-d6).

A currently accepted classification system for solvates and hydrates oforganic compounds is one that distinguishes between isolated site,channel, and metal-ion coordinated solvates and hydrates. See, e.g., K.R. Morris (H. G. Brittain ed.) Polymorphism in Pharmaceutical Solids(1995). Isolated site solvates and hydrates are ones in which thesolvent (e.g., water) molecules are isolated from direct contact witheach other by intervening molecules of the organic compound. In channelsolvates, the solvent molecules lie in lattice channels where they arenext to other solvent molecules. In metal-ion coordinated solvates, thesolvent molecules are bonded to the metal ion.

When the solvent or water is tightly bound, the complex will have awell-defined stoichiometry independent of humidity. When, however, thesolvent or water is weakly bound, as in channel solvates and inhygroscopic compounds, the water or solvent content will depend onhumidity and drying conditions. v In such cases, non-stoichiometry willtypically be observed.

Compounds of formula I include imine, carbinolamine and carbinolamineether forms of the PDD. The carbinolamine or the carbinolamine ether isformed when a nucleophilic solvent (H₂O, ROH) adds across the imine bondof the PDD moiety. The balance of these equilibria between these formsdepend on the conditions in which the compounds are found, as well asthe nature of the moiety itself.

These compounds may be isolated in solid form, for example, bylyophilisation.

Further particular and preferred aspects are set out in the accompanyingindependent and dependent claims. Features of the dependent claims maybe combined with features of the independent claims as appropriate, andin combinations other than those explicitly set out in the claims.

Synthetic Strategies

The compounds of Formula (I) may be prepared using the techniquesdescribed below. Some of the schemes and examples may omit details ofcommon reactions, including oxidations, reductions, and so on,separation techniques (extraction, evaporation, precipitation,chromatography, filtration, trituration, crystallization, and the like),and analytical procedures, which are known to persons of ordinary skillin the art of organic chemistry. The details of such reactions andtechniques can be found in a number of treatises, including RichardLarock, Comprehensive Organic Transformations, A Guide to FunctionalGroup Preparations, 2nd Ed (2010), and the multi-volume series edited byMichael B. Smith and others, Compendium of Organic Synthetic Methods(1974 et seq.). Starting materials and reagents may be obtained fromcommercial sources or may be prepared using literature methods. Some ofthe reaction schemes may omit minor products resulting from chemicaltransformations (e.g., an alcohol from the hydrolysis of an ester, CO₂from the decarboxylation of a diacid, etc.). In addition, in someinstances, reaction intermediates may be used in subsequent stepswithout isolation or purification (i.e., in situ).

In some of the reaction schemes and examples below, certain compoundscan be prepared using protecting groups, which prevent undesirablechemical reaction at otherwise reactive sites. Protecting groups mayalso be used to enhance solubility or otherwise modify physicalproperties of a compound. For a discussion of protecting groupstrategies, a description of materials and methods for installing andremoving protecting groups, and a compilation of useful protectinggroups for common functional groups, including amines, carboxylic acids,alcohols, ketones, aldehydes, and so on, see T. W. Greene and P. G.Wuts, Protecting Groups in Organic Chemistry, 4th Edition, (2006) and P.Kocienski, Protective Groups, 3rd Edition (2005).

Generally, the chemical transformations described throughout thespecification may be carried out using substantially stoichiometricamounts of reactants, though certain reactions may benefit from using anexcess of one or more of the reactants. Additionally, many of thereactions disclosed throughout the specification may be carried out atabout room temperature (RT) and ambient pressure, but depending onreaction kinetics, yields, and so on, some reactions may be run atelevated pressures or employ higher temperatures (e.g., refluxconditions) or lower temperatures (e.g., −78° C. to 0° C.). Anyreference in the disclosure to a stoichiometric range, a temperaturerange, a pH range, etc., whether or not expressly using the word“range,” also includes the indicated endpoints.

Many of the chemical transformations may also employ one or morecompatible solvents, which may influence the reaction rate and yield.Depending on the nature of the reactants, the one or more solvents maybe polar protic solvents (including water), polar aprotic solvents,non-polar solvents, or some combination. Representative solvents includesaturated aliphatic hydrocarbons (e.g., n-pentane, n-hexane, n-heptane,n-octane); aromatic hydrocarbons (e.g., benzene, toluene, xylenes);halogenated hydrocarbons (e.g., methylene chloride, chloroform, carbontetrachloride); aliphatic alcohols (e.g., methanol, ethanol,propan-1-ol, propan-2-ol, butan-1-ol, 2-methyl-propan-1-ol, butan-2-ol,2-methyl-propan-2-ol, pentan-1-ol, 3-methyl-butan-1-ol, hexan-1-ol,2-methoxy-ethanol, 2-ethoxy-ethanol, 2-butoxy-ethanol,2-(2-methoxy-ethoxy)-ethanol, 2-(2-ethoxy-ethoxy)-ethanol,2-(2-butoxy-ethoxy)-ethanol); ethers (e.g., diethyl ether, di-isopropylether, dibutyl ether, 1,2-dimethoxy-ethane, 1,2-diethoxy-ethane,1-methoxy-2-(2-methoxy-ethoxy)-ethane,1-ethoxy-2-(2-ethoxy-ethoxy)-ethane, tetrahydrofuran, 1,4-dioxane);ketones (e.g., acetone, methyl ethyl ketone); esters (methyl acetate,ethyl acetate); nitrogen-containing solvents (e.g., formamide,N,N—dimethylformamide, acetonitrile, N—methyl-pyrrolidone, pyridine,quinoline, nitrobenzene); sulfur-containing solvents (e.g., carbondisulfide, dimethyl sulfoxide, tetrahydro-thiophene-1,1-dioxide); andphosphorus-containing solvents (e.g., hexamethylphosphoric triamide).

Further particular and preferred aspects are set out in the accompanyingindependent and dependent claims. Features of the dependent claims maybe combined with features of the independent claims as appropriate, andin combinations other than those explicitly set out in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described further, withreference to the accompanying drawings, in which:

FIG. 1 shows a HPLC chromatogram that provides evidence of DNA adductformation with NFκB transcription factor binding sequence with C8-linkedPDD monomer 13.

FIG. 2 shows a HPLC chromatogram that provides evidence of DNA adductformation with NFκB transcription factor binding sequence with C8-linkedPDD monomer 17.

FIG. 3 shows a HPLC chromatogram that provides evidence of DNA adductformation with NFκB transcription factor binding sequence with C8-linkedPDD monomer 20.

FIG. 4 shows a HPLC chromatogram that provides evidence of DNA adductformation with NFκB transcription factor binding sequence with C8-linkedPDD monomer 24.

FIG. 5 shows the sequence of the labelled strand of the TyrT DNAfragment used in the cross-linking study.

FIG. 6 shows the autoradiograph of a denaturing polyacrylamide gelinvestigating the mechanism of DNA interaction of 41 with linear³²P-end-labelled TyrT DNA following overnight incubation at 37° C. atvarious concentrations.

FIG. 7 shows an autoradiograph of a denaturing polyacrylamide gelshowing DNA interstrand cross-linking by the PBD dimer Talirine withlinear ³²P-end-labelled TyrT DNA following overnight incubation at 37°C. at various concentrations.

FIG. 8A shows DNA footprint illustrating the interaction of 73 (B), 76(D), 81 (E), 88 (C) and 93 (G) with the MS1 DNA fragment. Ligandconcentrations are shown at the top of the gel. Tracks labelled “GA” aremarkers for specific purines.

FIG. 8B shows DNA footprint illustrating the interaction of 73 (B), 76(D), 81 (E), 88 (C) and 93 (G) with the HexA DNA fragment. Ligandconcentrations are shown at the top of the gel. Tracks labelled “GA” aremarkers for specific purines.

FIG. 8C shows DNA footprint illustrating the interaction of the PBDdimer Talirine with the MS1 DNA fragment. Ligand concentrations areshown at the top of the gel. Tracks labelled “GA” are markers forspecific purines.

FIG. 9 shows the sequence of the MS1 DNA fragment showing the possiblemono-alkylated adducts produced by the compounds analysed.

FIG. 10 shows the sequence of the HexA DNA fragment showing the possiblemono-alkylated adducts produced by the compounds analysed.

FIG. 11 shows fluorescently labelled DNA duplex used in the FRET meltingstudy to study the stabilisation of DNA by 41, 106, 107 and 148. Thelabels were fluorescein (F) and dabcyl (Q).

FIG. 12A shows FRET denaturation data for 5′-AAAAAAAGAAATTTAAA-3′ whenbound to 41.

FIG. 12B shows FRET denaturation data for 5′-AAAAAAAGAAATTTAAA-3′ whenbound to 106, 148 and 107 (right to left).

FIG. 13A shows a graph illustrating percentage difference in TFactivation in cells treated with compound 41.

FIG. 13B shows a graph illustrating percentage difference in TFactivation in cells not treated with 41.

FIG. 14 shows a graph illustrating a summary of the major transcriptionfactors up- and down-regulated by 41.

FIG. 15A shows a graph illustrating cell cycle arrest by 41.

FIG. 15B shows a graph illustrating cell cycle arrest by Talirine.

FIG. 16 shows a graph outlining cell cycle arrest induced by 41.

FIG. 17 shows an SEC profile of Antibody X.

FIG. 18 shows an HIC profile of Antibody X.

FIG. 19 shows a PLRP trace of Antibody X. Heavy (H0) and light (L0)chain peaks as indicated.

FIG. 20 shows an HIC profile of IgG1-141. Average DAR calculated as 1.9with the DAR species assignments as indicated.

FIG. 21 shows a PLRP trace of IgG1-141. Average DAR calculated as 1.8with the light/heavy chain species assigned as indicated.

FIG. 22 shows an SEC profile of IgG1-141; 96.5% monomer, 3.4% dimer,0.1% HMW as indicated.

FIG. 23 shows the free toxin linker traces of the IgG1-141 sample. Nofree toxin linker could be detected in the ADC trace. Red: 100 pmolNAC-141. Blue: IgG1-141 after protein precipitation; the identifiedpeaks show residual proteinaceous material. Green: PBS.

FIG. 24A shows an example of HIC analysis used to assign DAR to thetrastuzumab-based ADC.

FIG. 24B shows an example of SEC analysis used to assign DAR to thetrastuzumab-based ADC.

FIG. 25 shows an SEC profile of the THIOMAB. 92.4% monomer, 6.7% dimer,and 0.9% HMW as indicated. The peak at about 23 minutes originates fromthe formulation of the antibody;

FIG. 26 shows an HIC profile of the THIOMAB®-based Trastuzumab.

FIG. 27 shows the reducing-PLRP trace of the THIOMAB. Heavy (H0) andlight (L0) chain peaks as indicated.

FIG. 28 shows the HIC profile of THIOMAB-141. Average DAR calculated as1.9 with the DAR species assignments as indicated.

FIG. 29 shows the PLRP trace of THIOMAB-141. Average DAR calculated as1.8 with the light/heavy chain species assigned as indicated.

FIG. 30 shows the SEC profile of THIOMAB-141; 94.8% monomer, 4.8% dimer,0.4% HMW as indicated.

FIG. 31 shows a graph illustrating dose tolerability of 141, PBD dimer(Talirine) and MMAE-based ADCs.

FIG. 32 shows a graph illustrating mean tumour volume versus time aftertwo doses of ADC 2 (Day 0 and Day 7).

FIG. 33 shows a graph illustrating mean tumour volume of a PDX modelversus time after two doses of ADC 2 (Day 0 and Day 14).

FIG. 34A shows an example of a compound of the disclosure attached tolinker group containing an exemplary terminal alkoxyamine groups

FIG. 34B shows an example of a compound of the disclosure attached tolinker group containing an exemplary terminal diarylcyclooctyne group,DBCO.

EXAMPLES

General Remarks

Reagents were purchased from standard commercial suppliers. Solventswere purchased from Sigma-Aldrich (UK) and Fisher Scientific (UK).Anhydrous reactions were carried out in pre-oven-dried glassware underan inert atmosphere of nitrogen. Anhydrous solvents were used aspurchased without further drying. Thin Layer Chromatography (TLC) wasperformed on silica gel aluminium plates (Merck 60, F₂₅₄), and columnchromatography was carried out either manually using silica gel (Merck9385, 230-400 mesh ASTM, 40-63 μM) (whilst monitoring by thin layerchromatography: UV (254 nm) and an aqueous alkaline solution ofpotassium permanganate as stain), or using a Biotage Isolera One. AllNMR spectra were obtained at room temperature using a Bruker DPX400spectrometer, for which chemical shifts are expressed in ppm relative tothe solvent and coupling constants are expressed in Hz. All LiquidChromatography Mass Spectroscopy (LCMS) analysis was performed on aWaters Alliance 2695 with water (A) and acetonitrile (B) comprising themobile phases. Formic acid (0.1%) was added to the acetonitrile toensure acidic conditions throughout the analysis. Function type: Diodearray (535 scans). Column type: Monolithic C18 50×4.60 mm. Massspectrometry data were collected using a Waters Micromass ZQ instrumentcoupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters MicromassZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor(V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (°C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250.Microwave reactions were carried out on a Biotage Initiator+microwavesynthesis reactor. HRMS was performed on a Thermo Scientific-ExactiveHCD Orbitrap Mass Spectrometer. Yields refer to isolated material(homogeneous by TLC or NMR) unless otherwise stated and names areassigned according to IUPAC nomenclature. LCMS gradient conditions aredescribed as follows.

Method A (10 min): from 95% A/5% B to 50% B over 3 min. Then from 50% Bto 80% B over 2 min. Then from 80% B to 95% B over 1.5 min and heldconstant for 1.5 min. This was then reduced to 5% B over 0.2 min andmaintained to 5% B for 1.8 min. The flow rate was 0.5 mL/min, 200 μL wassplit via a zero dead volume T piece which passed into the massspectrometer. The wavelength range of the UV detector was 220-400 nm.

Method B (5 min): from 95% A/5% B to 90% B over 3 min. Then from 90% Bto 95% B over 0.5 min and held constant for 1 min. This was then reducedto 5% B over 0.5 min. The flow rate was 1.0 mL/min, 100 μL was split viaa zero dead volume T piece which passed into the mass spectrometer. Thewavelength range of the UV detector was 220-500 nm.

General Synthetic Scheme

Example 1

Methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (1)

A mixture of vanillin (20.0 g, 131 mmol), methyl 4-bromobutanoate (175mL, 139 mmol) and potassium carbonate (27.2 g, 197 mmol) inN,N—dimethylformamide (100 mL) was stirred at room temperature for 18 h.The reaction mixture was diluted with water (500 mL) and the titlecompound (30.2 g, 91%) was obtained by filtration as a white solid. Theproduct was carried through to the next step without any furtherpurification.

¹H NMR (400 MHz, CDCl₃) δ 9.84 (s, 1H), 7.46-7.37 (m, 2H), 6.98 (d,J=8.2 Hz, 1H), 4.16 (t, J=6.3 Hz, 2H), 3.91 (s, 3H), 3.69 (s, 3H), 2.56(t, J=7.2 Hz, 2H), 2.20 (quin, J=6.7 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ190.9, 173.4, 153.8, 149.9, 130.1, 126.8, 111.6, 109.2, 67.8, 56.0,51.7, 30.3, 24.2; MS m/z (EIMS)=271.9 (M+Na)⁺; LCMS (Method A):t_(R)=6.48 min.

Example 2

Methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (2)

To a stirring solution of potassium nitrate (10.0 g, 98.9 mmol) in TFA(50 mL) at 0° C. was added dropwise a solution of methyl4-(4-formyl-2-methoxyphenoxy)butanoate (1) (20.0 g, 79.2 mmol) in TFA(50 mL). The reaction mixture was stirred at room temperature for 1 h.It was then concentrated in vacuo and diluted with ethyl acetate (400mL). The organic layer was washed with brine (3×100 mL) and a saturatedaqueous solution of sodium hydrogen carbonate (2×80 mL), dried oversodium sulfate, filtered and concentrated to give the title compound(23.5 g, 100%) as a yellow solid. The product was carried through to thenext step without any further purification.

¹H NMR (400 MHz, CDCl₃) δ 10.42 (s, 1H), 7.60 (s, 1H), 7.39 (s, 1H),4.21 (t, J=6.3 Hz, 2H), 3.98 (s, 3H), 3.70 (s, 3H), 2.61-2.53 (m, 2H),2.22 (quin, J=6.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 187.8, 173.2,153.5, 151.7, 143.8, 125.5, 109.9, 108.1, 68.6, 56.6, 51.8, 30.2, 24.1;MS m/z (EIMS)=296.1 (M−H)⁻; LCMS (Method A): t_(R=6.97) min.

Example 3

5-Methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic Acid (3)

To a solution of methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate(2) (23.0 g, 77.4 mmol) in acetone (600 mL) was quickly added a hot (70°C.) solution of potassium permanganate (46.0 g, 291 mmol) in water (400mL). The reaction mixture was stirred at 70° C. for 3 h. The reactionmixture was cooled to room temperature and passed through celite. Thecake of celite was washed with hot water (200 mL). A solution of sodiumbisulfite in hydrochloric acid (1 M, 200 mL) was added to the filtratewhich was extracted with dichloromethane (2×400 mL). The organic layerwas dried over sodium sulfate, filtered and concentrated. The resultingresidue was purified by column chromatography (silica), eluting withmethanol/dichloromethane (from 0% to 50%), to give the title compound(17.0 g, 70%) as a pale yellow solid.

¹H NMR (400 MHz, MeOD) δ 7.47 (s, 1H), 7.25 (s, 1H), 4.13 (t, J=6.2 Hz,2H), 3.94 (s, 3H), 3.68 (s, 3H), 2.54 (t, J=7.2 Hz, 2H), 2.17-2.06 (m,2H); ¹³C NMR (100 MHz, MeOD) δ 175.3, 168.6, 153.8, 151.3, 143.1, 122.8,112.4, 109.2, 69.6, 57.0, 52.2, 31.2, 25.5; MS m/z (EIMS)=311.9 (M−H)⁻;LCMS (Method A): t_(R=6.22) min.

Example 4

Methyl(S)—4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)butanoate(4)

A mixture of 5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic acid (3)(8.0 g, 25.5 mmol), oxalyl chloride (6.6 mL, 77.0 mmol) and anhydrousN,N—dimethl-formamide (2 drops) in anhydrous dichloromethane (100 mL)was stirred at room temperature for 1 h. Anhydrous toluene (20 mL) wasadded to the reaction mixture which was then concentrated in vacuo. Asolution of the resulting residue in anhydrous dichloromethane (10 mL)was added dropwise to a solution of (S)—piperidin-2-ylmethanol (3.8 g,33.4 mmol) and triethylamine (10.7 mL, 77.0 mmol) in anhydrousdichloromethane (90 mL) at −10° C. The reaction mixture was stirred atroom temperature for 2 h and then washed with hydrochloric acid (1 M, 50mL) and a saturated aqueous solution of sodium chloride (50 mL), driedover sodium sulfate, filtered and concentrated. The resulting residuewas purified by column chromatography (silica), eluting withmethanol/dichloromethane (from 0% to 5%), to give the title compound(9.2 g, 73%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 7.68-7.64 (m, 1H), 6.77-6.70 (m, 1H),4.16-4.07 (m, 3H), 3.93-3.89 (m, 3H), 3.83 (s, 1H), 3.67 (s, 3H), 3.15(d, J=1.4 Hz, 1H), 3.11 (s, 1H), 2.78 (s, 1H), 2.56-2.50 (m, 3H),2.21-2.12 (m, 4H), 1.74-1.55 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 173.3,168.1, 154.6, 148.2, 137.4, 127.6, 111.4, 108.3, 68.3, 60.6, 56.7, 53.5,51.7, 43.3, 38.0, 34.9, 30.3, 24.1, 19.7; MS m/z (EIMS)=411.0 (M+H)⁺;LCMS (Method A): t_(R=6.28) min.

Example 5

Methyl(S)—4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate(5)

To a solution of methyl(S)—4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)butanoate(4) (9.2 g, 22.4 mmol) in ethanol (40 mL) and ethyl acetate (10 mL) wasadded palladium on activated charcoal (10% wt. basis) (920 mg). Thereaction mixture was hydrogenated at 35 psi for 3 h in a Parr apparatus.The reaction mixture was filtered through celite and the resulting cakewas washed with ethyl acetate. The filtrate was concentrated in vacuo togive the title compound (9.0 g, 90%) as a pink solid. The product wascarried through to the next step without any further purification.

¹H NMR (400 MHz, CDCl₃) δ 6.69 (s, 1H), 6.27-6.18 (m, 1H), 4.03-3.94 (m,3H), 3.94-3.82 (m, 3H), 3.81-3.76 (m, 1H), 3.74 (s, 3H), 3.73-3.68 (m,1H), 3.67-3.65 (m, 3H), 3.56 (d, J=4.8 Hz, 1H), 3.03 (s, 1H), 2.51 (t,J=7.2 Hz, 2H), 2.11 (quin, J=6.7 Hz, 2H), 1.68-1.59 (m, 4H), 1.55-1.40(m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.6, 171.2, 150.3, 141.8, 141.1,113.2, 112.3, 102.4, 67.5, 60.8, 60.4, 56.8, 51.6, 30.4, 25.8, 24.3,21.0, 19.9, 14.2; MS m/z (EIMS)=381.0 (M+H)⁺; LCMS (Method A):t_(R=5.52) min.

Example 6

Methyl(S)—4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxyl-methyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate(6)

To a solution of methyl(S)—4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl-2-methoxyphenoxy)butanoate(5) (9.0 g, 23.7 mmol) and pyridine (4.4 mL, 54.4 mmol) in anhydrousdichloromethane (100 mL) at −10° C. was added dropwise a solution ofallylchloroformate (2.6 mL, 24.8 mmol) in anhydrous dichloromethane (20mL). The reaction mixture was stirred at room temperature for 30 min.The reaction mixture was sequentially washed with a saturated aqueoussolution of copper (II) sulfate (80 mL), water (80 mL) and a saturatedaqueous solution of sodium hydrogen carbonate (80 mL). The organic layerwas dried over sodium sulfate, filtered and concentrated. The resultingresidue (2.0 g out of the 11.0 g crude) was purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 1%), to give the title compound (930 mg, 47% based on the amountpurified) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 8.30 (br s, 1H), 7.63 (br s, 1H), 6.76 (br s,1H), 5.92 (ddt, J=17.2, 10.6, 5.4, 5.4 Hz, 1H), 5.37-5.28 (m, 1H), 5.20(dq, J=10.4, 1.3 Hz, 1H), 4.65-4.56 (m, 2H), 4.06 (t, J=6.2 Hz, 2H),3.94-3.82 (m, 1H), 3.79 (s, 3H), 3.66 (s, 3H), 3.62-3.54 (m, 1H), 3.40(br s, 1H), 3.10-2.88 (m, 1H), 2.52 (t, J=7.4 Hz, 2H), 2.22-2.04 (m,3H), 1.64 (br s, 4H), 1.56-1.31 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ173.5, 170.6, 153.9, 149.7, 144.8, 132.6, 130.1, 117.6, 116.9, 110.8,107.1, 106.0, 67.7, 65.6, 60.7, 56.3, 53.5, 51.6, 43.1, 30.5, 25.7,24.4, 19.7; MS m/z (EIMS)=465.1 (M+H)⁺; LCMS (Method A): t_(R=6.47) min.

Example 7

Allyl(6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(7)

To a solution of methyl(S)—4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)-piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate(6) (930 mg, 2.0 mmol) in dichloromethane (45 mL) was added TEMPO (32mg, 0.20 mmol) and (diacetoxyiodo)-benzene (773 mg, 2.4 mmol). Thereaction mixture was stirred at room temperature for 16 h, and was thensequentially washed with a saturated aqueous solution of sodiummetabisulfite (20 mL), a saturated aqueous solution of sodium hydrogencarbonate (20 mL), water (20 mL) and brine (20 mL). The organic layerwas then dried over sodium sulfate, filtered and concentrated. Theresulting residue was purified by column chromatography (silica),eluting with methanol/dichloromethane (from 0% to 5%), to give the titlecompound (825 mg, 89%) as a cream solid.

MS m/z (EIMS)=462.9 (M+H)⁺; LCMS (Method A): t_(R=6.30) min.

Example 8

Allyl(6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepine-5(12H)-carboxylate(8)

A mixture of allyl(6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(7) (825 mg, 1.8 mmol), 3,4-dihydro-2H-pyran (1.7 mL, 18.2 mmol) andpTSA (8.5 mg, 1% w/w) in ethyl acetate (12 mL) was stirred at roomtemperature for 16 h. The reaction mixture was then diluted with ethylacetate (50 mL) and washed with a saturated aqueous solution of sodiumhydrogen carbonate (20 mL) and brine (30 mL). The organic layer wasdried over sodium sulfate, filtered and concentrated. The resultingresidue was purified by column chromatography (silica), eluting withmethanol/dichloromethane (from 0% to 2%), to give the title compound(820 mg, 84%) as a cream solid.

MS m/z (EIMS)=546.7 (M+H)⁺; LCMS (Method A): t_(R=7.70) min.

Example 9

4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicAcid (9)

To a solution of allyl(6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(8) (770 mg, 1.4 mmol) in 1,4-dioxane (10 mL) was added a 0.5 M aqueoussolution of sodium hydroxide (10 mL, 5.0 mmol). The reaction mixture wasstirred at room temperature for 2 h and was then concentrated in vacuo,after which water (20 mL) was added and the aqueous layer was acidifiedto pH=1 with a 1 M citric acid solution (5 mL). The aqueous layer wasthen extracted with ethyl acetate (2×50 mL). The combined organicextracts were then washed with brine (50 mL), dried over sodium sulfate,filtered and concentrated to give the title compound (700 mg, 93%) as ayellow oil. The product was carried through to the next step without anyfurther purification.

MS m/z (EIMS)=532.9 (M+H)⁺; LCMS (Method A): t_(R=6.98) min.

Example 10

Methyl5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylate(10)

A solution of4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid(500 mg, 2.1 mmol) in N,N—dimethylformamide (10 mL) was charged with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (725 mg, 3.8mmol) and 4-(dimethylamino)pyridine (577 mg, 4.7 mmol). The reactionmixture was stirred at room temperature for 2 h. Methyl5-aminobenzo[b]thiophene-2-carboxylate (392 mg, 1.9 mmol) was then addedand the resulting mixture was stirred at room temperature for 16 h. Thiswas then poured into ice-water (20 mL) and extracted with ethyl acetate(3×50 mL). The combined organic extracts were sequentially washed with 1M citric acid (30 mL), a saturated aqueous solution of sodium hydrogencarbonate (35 mL), water (35 mL) and brine (35 mL). The organic layerwas then dried over sodium sulfate, filtered and concentrated. Theresulting residue was purified by column chromatography (silica),eluting with ethyl acetate/hexane (from 0% to 50%), to give the titlecompound (610 mg, 75%) as a beige solid.

MS m/z (EIMS)=430.2 (M+H)⁺; LCMS (Method A): t_(R=7.90) min.

Example 11

Methyl5-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)-benzo[b]thiophene-2-carboxylateHydrochloride (11)

Methyl5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzo-[b]thiophene-2-carboxylate(10) (610 mg, 1.4 mmol) was dissolved in hydrochloric acid (4 M indioxane) (3.6 mL) and the reaction mixture was stirred at roomtemperature for 2 h. The reaction mixture was concentrated in vacuo togive the title compound (600 mg, 99%) as a brown solid. The product wascarried through to the next step without any further purification.

MS m/z (EIMS)=329.9 (M+H)⁺; LCMS (Method A): t_(R=5.52) min.

Example 12

Allyl(6aS)-2-methoxy-3-(4-((5-((2-(methoxycarbonyl)benzo-[b]thiophen-5-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(12)

A solution of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (150 mg, 0.28 mmol) in N,N—dimethylformamide (4 mL) was chargedwith 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100mg, 0.52 mmol) and 4-(dimethylamino)pyridine (80 mg, 0.65 mmol). Thereaction mixture was stirred at room temperature for 30 min. Methyl5-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylatehydrochloride (11) (95 mg, 0.26 mmol) was then added and the resultingmixture was stirred at room temperature for 16 h. This was then pouredinto ice-water (20 mL) and extracted with ethyl acetate (3×50 mL). Thecombined organic extracts were sequentially washed with 1 M citric acid(30 mL), a saturated aqueous solution of sodium hydrogen carbonate (35mL), water (35 mL) and brine (35 mL). The organic layer was then driedover sodium sulfate, filtered and concentrated in vacuo to give thetitle compound (190 mg, 87%) as a yellow oil. The product was carriedthrough to the next step without any further purification.

MS m/z (EIMS)=844.0 (M+H)⁺; LCMS (Method A): t_(R=8.10) min.

Example 13

Methyl(S)—5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylate(13)

To a solution of allyl(6aS)-2-methoxy-3-(4-((5-((2-(methoxycarbonyl)benzo[b]-thiophen-5-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate(12) (190 mg, 0.22 mmol) in dichloromethane (10 mL) was addedtetrakis(triphenylphosphine)palladium(0) (13 mg, 5 mol %),triphenylphosphine (15 mg, 25 mol %) and pyrrolidine (22 μL, 0.27 mmol).The reaction mixture was stirred at room temperature for 30 min. Thereaction mixture was subjected to high vacuum for 30 min until excesspyrrolidine was thoroughly removed. The resulting residue was thenpurified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 70%), to give the title compound (60mg, 40%) as a yellow solid.

¹H NMR (CDCl₃, 400 MHz) δ 8.35 (s, 1H), 8.28 (s, 1H), 8.02 (s, 1H), 7.94(s, 1H), 7.90 (d, J=5.7 Hz, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.58 (dd,J=8.7, 2.1 Hz, 1H), 7.42-7.41 (m, 1H), 7.13 (d, J=1.6 Hz, 1H), 6.78 (s,1H), 6.56 (d, J=1.6 Hz, 1H), 4.25-4.18 (m, 1H), 4.08 (t, J=6.0 Hz, 2H),3.93 (s, 3H), 3.88 (s, 3H), 3.83 (s, 3H), 3.79-3.75 (m, 1H), 3.23-3.16(m, 1H), 2.52-2.47 (m, 2H), 2.21 (d, J=6.4 Hz, 1H), 2.18 (d, J=2.1 Hz,1H), 1.96 (br s, 2H), 1.86-1.81 (m, 2H), 1.77-1.66 (m, 2H); ¹³C NMR (100MHz, CDCl₃) δ 170.0, 167.6, 163.4, 163.2, 160.0, 150.7, 148.0, 140.0,139.2, 137.6, 135.8, 134.2, 130.6, 123.0, 122.9, 121.5, 121.0, 120.1,116.2, 111.7, 110.3, 104.3, 68.1, 56.1, 53.5, 52.5, 49.7, 40.0, 36.8,33.0, 24.9, 24.5, 22.9, 18.3; MS m/z (EIMS)=658.0 (M+H)⁺; LCMS (MethodA): t_(R=6.92) min.

Example 14

Allyl(6aS)-3-(4-((2-(ethoxycarbonyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)-oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4a]diazepine-5(12H)-carboxylate(14)

A solution of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (340 mg, 0.64 mmol) in N,N—dimethylformamide (10 mL) wascharged with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(222 mg, 1.2 mmol) and 4-(dimethylamino)pyridine (177 mg, 1.4 mmol). Thereaction mixture was stirred at room temperature for 30 min. Ethyl4-amino-1-methyl-1H-imidazole-2-carboxylate hydrochloride (120 mg, 0.58mmol) was then added and the resulting mixture was stirred at roomtemperature for 16 h. This was then poured into ice-water (40 mL) andextracted with ethyl acetate (3×100 mL). The combined organic extractswere sequentially washed with 1 M citric acid (60 mL), a saturatedaqueous solution of sodium hydrogen carbonate (70 mL), water (70 mL) andbrine (70 mL). The organic layer was then dried over sodium sulfate,filtered and concentrated in vacuo to give the title compound (350 mg,80%) as a yellow oil. The product was carried through to the next stepwithout any further purification.

MS m/z (EIMS)=683.7 (M+H)⁺; LCMS (Method A): t_(R=7.35) min.

Example 15

4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxylicAcid (15)

To a solution of allyl(6aS)-3-(4-((2-(ethoxycarbonyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(14) (350 mg, 0.46 mmol) in 1,4-dioxane (10 mL) was added a 0.5 Maqueous solution of sodium hydroxide (10 mL, 5.0 mmol). The reactionmixture was stirred at room temperature for 2 h and was thenconcentrated in vacuo, after which water (20 mL) was added and theaqueous layer was acidified to pH=1 with a 1 M citric acid solution (10mL). The aqueous layer was then extracted with ethyl acetate (2×50 mL).The combined organic extracts were then washed with a saturated aqueoussolution of sodium chloride (50 mL), dried over sodium sulfate, filteredand concentrated. The resulting residue was triturated in hexane,filtered and dried to give the title compound (220 mg, 74%) as a beigesolid. The product was carried through to the next step without anyfurther purification.

MS m/z (EIMS)=656.2 (M+H)⁺; LCMS (Method A): t_(R=6.53) min.

Example 16

Allyl(6aS)-2-methoxy-3-(4-((2-((2-(methoxycarbonyl)-benzo[b]thiophen-5-yl)carbamoyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(16)

A solution of4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxylicacid (15) (110 mg, 0.17 mmol) in N,N—dimethylformamide (4 mL) wascharged with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(59 mg, 0.31 mmol) and 4-(dimethylamino)pyridine (47 mg, 0.38 mmol). Thereaction mixture was stirred at room temperature for 30 min. Methyl5-aminobenzo[b]thiophene-2-carboxylate (32 mg, 0.15 mmol) was then addedand the resulting mixture was stirred at room temperature for 16 h. Thiswas then poured into ice-water (40 mL) and extracted with ethyl acetate(3×100 mL). The combined organic extracts were sequentially washed with1 M citric acid (60 mL), a saturated aqueous solution of sodium hydrogencarbonate (70 mL), water (70 mL) and brine (70 mL). The organic layerwas then dried over sodium sulfate, filtered and concentrated. Theresulting residue was then purified by column chromatography (silica),eluting with ethyl acetate/dichloromethane (0% to 100%), followed bymethanol/dichloromethane (from 0% to 10%), to give the title compound(50 mg, 39%) as a yellow oil.

MS m/z (EIMS)=844.9 (M+H)⁺; LCMS (Method A): t_(R=8.22) min.

Example 17

Methyl(S)—5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[b]thiophene-2-carboxylate(17)

To a solution of allyl(6aS)-2-methoxy-3-(4-((2-((2-(methoxycarbonyl)benzo[b]-thiophen-5-yl)carbamoyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate(16) (50 mg, 0.06 mmol) in dichloromethane (3 mL) was addedtetrakis(triphenylphosphine)palladium(0) (3.5 mg, 5 mol %),triphenylphosphine (3.9 mg, 25 mol %) and pyrrolidine (5.8 μL, 0.07mmol). The reaction mixture was stirred at room temperature for 30 min.The reaction mixture was subjected to high vacuum for 30 min untilexcess pyrrolidine was thoroughly removed. The resulting residue wasthen purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 50%), to give the title compound (10mg, 26%) as a yellow solid.

¹H NMR (CDCl₃, 400 MHz) 9.07 (s, 1H), 8.36 (d, J=2.0 Hz, 1H), 8.13 (s,1H), 8.03 (s, 1H), 7.90 (d, J=5.7 Hz, 1H), 7.82 (d, J=8.7 Hz, 1H), 7.56(dd, J=8.7, 2.1 Hz, 1H), 7.49-7.43 (m, 2H), 6.81 (s, 1H), 4.26-4.17 (m,2H), 4.10-4.06 (m, 3H), 3.98-3.93 (m, 6H), 3.93-3.85 (m, 1H), 3.74 (td,J=5.8, 4.0 Hz, 1H), 3.27-3.16 (m, 1H), 2.68-2.60 (m, 2H), 2.29 (quin,J=6.4 Hz, 2H), 2.10-2.02 (m, 1H), 1.97-1.89 (m, 1H), 1.83-1.77 (m, 2H),1.76 (s, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 169.7, 167.5, 163.3, 163.2,160.3, 156.7, 150.4, 148.0, 140.0, 139.3, 135.8, 135.0, 130.6, 123.2,120.1, 115.4, 114.9, 110.3, 98.0, 67.8, 65.2, 56.1, 52.6, 49.6, 39.8,35.9, 32.9, 31.0, 29.3, 24.7, 24.6, 22.9, 18.4; MS m/z (EIMS)=659.1(M+H)⁺; LCMS (Method A): t_(R=7.00) min.

Example 18

Methyl 4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (18)

A mixture of methyl 4-bromo-1-methyl-1H-pyrrole-2-carboxylate (750 mg,3.44 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (905mg, 4.13 mmol) and potassium carbonate (1.43 g, 10.3 mmol) intoluene/ethanol/water (9:3:1) (13 mL total) was degassed with nitrogenfor 5 mins. Tetrakis(triphenylphosphine)palladium(0) (230 mg, 6 mol %)was then charged and the reaction mixture was irradiated with microwavesat 100° C. for 15 mins. Water (10 mL) was then added to the reactionmixture, which was extracted with ethyl acetate (3×40 mL). The combinedorganic extracts were then dried over sodium sulfate, filtered andconcentrated. The resulting residue was purified by columnchromatography (silica), eluting with ethyl acetate/hexanes (from 0% to50%), to give the title compound (145 mg, 18%) as a yellow solid.

MS m/z (EIMS)=230.9 (M+H)⁺; LCMS (Method A): t_(R=5.17) min.

Example 19

Allyl(6S,6aS)-2-methoxy-3-(4-((2-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(19)

A solution of4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxylicacid (15) (110 mg, 0.17 mmol) in N,N—dimethylformamide (4 mL) wascharged with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(59 mg, 0.31 mmol) and 4-(dimethylamino)pyridine (47 mg, 0.38 mmol). Thereaction mixture was stirred at room temperature for 30 min. Methyl4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (18) (35 mg, 0.15mmol) was then added and the resulting mixture was stirred at roomtemperature for 16 h. This was then poured into ice-water (40 mL) andextracted with ethyl acetate (3×100 mL). The combined organic extractswere sequentially washed with 1 M citric acid (60 mL), a saturatedaqueous solution of sodium hydrogen carbonate (70 mL), water (70 mL) andbrine (70 mL). The organic layer was then dried over sodium sulfate,filtered and concentrated. The resulting residue was then purified bycolumn chromatography (silica), eluting with acetone/dichloromethane (0%to 50%), to give the title compound (54 mg, 37%) as a yellow oil.

MS m/z (EIMS)=868.1 (M+H)⁺; LCMS (Method A): t_(R=8.22) min.

Example 20

Methyl(S)—4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(20)

To a solution of allyl(6S,6aS)-2-methoxy-3-(4-((2-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(19) (54 mg, 0.06 mmol) in dichloromethane (3 mL) was addedtetrakis(triphenylphosphine)palladium(0) (3.6 mg, 5 mol %),triphenylphosphine (4.1 mg, 25 mol %) and pyrrolidine (6.2 μL, 0.07mmol). The reaction mixture was stirred at room temperature for 30 min.The reaction mixture was subjected to high vacuum for 30 min untilexcess pyrrolidine was thoroughly removed. The resulting residue wasthen purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 50%), to give the title compound (22mg, 52%) as a yellow solid.

¹H NMR (CDCl₃, 400 MHz) δ 8.95 (s, 1H), 8.27 (s, 1H), 7.89 (d, J=5.7 Hz,1H), 7.59 (d, J=8.6 Hz, 2H), 7.47-7.41 (m, 4H), 7.19 (d, J=2.0 Hz, 1H),7.05 (d, J=1.9 Hz, 1H), 6.79 (s, 1H), 4.25-4.18 (m, 1H), 4.17-4.12 (m,1H), 4.12-4.06 (m, 1H), 4.04 (s, 3H), 3.95 (s, 3H), 3.93 (s, 3H), 3.84(s, 3H), 3.76-3.71 (m, 1H), 3.26-3.16 (m, 1H), 2.65-2.57 (m, 2H), 2.26(t, J=6.4 Hz, 2H), 2.09-2.01 (m, 2H), 1.96-1.89 (m, 1H), 1.85-1.77 (m,2H), 1.67 (dd, J=10.9, 5.5 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 169.7,167.5, 163.3, 161.7, 156.5, 150.4, 148.0, 140.0, 135.8, 135.6, 133.7,130.6, 126.1, 125.5, 123.1, 122.8, 120.0, 114.6, 111.6, 110.2, 67.8,56.1, 51.2, 49.6, 39.8, 37.0, 35.8, 32.8, 31.0, 29.7, 24.7, 24.5, 22.9,18.4; MS m/z (EIMS)=682.1 (M+H)⁺; LCMS (Method A): t_(R=7.03) min.

Example 21

Allyl(6aS)-2-methoxy-3-(4-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(21)

A solution of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (150 mg, 0.64 mmol) in N,N—dimethylformamide (4 mL) was chargedwith 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (98 mg,0.51 mmol) and 4-(dimethylamino)pyridine (79 mg, 0.64 mmol). Thereaction mixture was stirred at room temperature for 30 min. Methyl4-amino-1-methyl-1H-pyrrole-2-carboxylate hydrochloride (49 mg, 0.26mmol) was then added and the resulting mixture was stirred at roomtemperature for 16 h. This was then poured into ice-water (40 mL) andextracted with ethyl acetate (3×100 mL). The combined organic extractswere sequentially washed with 1 M citric acid (60 mL), a saturatedaqueous solution of sodium hydrogen carbonate (70 mL), water (70 mL) andbrine (70 mL). The organic layer was then dried over sodium sulfate,filtered and concentrated in vacuo to give the title compound (150 mg,88%) as a yellow oil. The product was carried through to the next stepwithout any further purification.

MS m/z (EIMS)=668.8 (M+H)⁺; LCMS (Method A): t_(R=7.42) min.

Example 22

4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxylicAcid (22)

To a solution of allyl(6aS)-2-methoxy-3-(4-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(21) (150 mg, 0.22 mmol) in 1,4-dioxane (5 mL) was added a 0.5 M aqueoussolution of sodium hydroxide (5 mL, 5.0 mmol). The reaction mixture wasstirred at room temperature for 2 h and was then concentrated in vacuo,after which water (20 mL) was added and the aqueous layer was acidifiedto pH=1 with a 1 M citric acid solution (10 mL). The aqueous layer wasthen extracted with ethyl acetate (2×50 mL). The combined organicextracts were then washed with brine (50 mL), dried over sodium sulfate,filtered and concentrated in vacuo to give the title compound (140 mg,90%) as a beige solid. The product was carried through to the next stepwithout any further purification.

MS m/z (EIMS)=677.0 (M+Na)⁺; LCMS (Method A): t_(R=6.92) min.

Example 23

Allyl(6S,6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(23)

A solution of4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxylicacid (22) (140 mg, 0.21 mmol) in N,N—dimethylformamide (4 mL) wascharged with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(74 mg, 0.39 mmol) and 4-(dimethylamino)pyridine (59 mg, 0.48 mmol). Thereaction mixture was stirred at room temperature for 30 min. Methyl4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (18) (45 mg, 0.19mmol) was then added and the resulting mixture was stirred at roomtemperature for 16 h. This was then poured into ice-water (40 mL) andextracted with ethyl acetate (3×100 mL). The combined organic extractswere sequentially washed with 1 M citric acid (60 mL), a saturatedaqueous solution of sodium hydrogen carbonate (70 mL), water (70 mL) andbrine (70 mL). The organic layer was then dried over sodium sulfate,filtered and concentrated. The resulting residue was then purified bycolumn chromatography (silica), eluting with acetone/dichloromethane (0%to 50%), to give the title compound (160 mg, 95%) as a yellow solid.

MS m/z (EIMS)=867.0 (M+H)⁺; LCMS (Method A): t_(R=8.10) min.

Example 24

Methyl(S)—4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(24)

To a solution of allyl(6S,6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(23) (80 mg, 0.09 mmol) in dichloromethane (3 mL) was addedtetrakis(triphenylphosphine)palladium(0) (5.3 mg, 5 mol %),triphenylphosphine (6.1 mg, 25 mol %) and pyrrolidine (9.1 μL, 0.11mmol). The reaction mixture was stirred at room temperature for 30 min.The reaction mixture was subjected to high vacuum for 30 min untilexcess pyrrolidine was thoroughly removed. The resulting residue wasthen purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 50%), to give the title compound (23mg, 37%) as a yellow solid.

¹H NMR (CDCl₃, 400 MHz) δ 8.09 (s, 1H), 8.04-8.01 (m, 1H), 7.90 (d,J=5.8 Hz, 1H), 7.58 (s, 1H), 7.56 (s, 1H), 7.44-7.40 (m, 3H), 7.18 (d,J=2.0 Hz, 1H), 7.12 (d, J=1.8 Hz, 1H), 7.04 (d, J=2.0 Hz, 1H), 6.78 (s,1H), 6.50 (d, J=1.9 Hz, 1H), 4.26-4.18 (m, 1H), 4.07 (t, J=6.0 Hz, 2H),3.94 (s, 3H), 3.87 (s, 3H), 3.84 (d, J=2.9 Hz, 6H), 3.76 (td, J=5.7, 3.9Hz, 1H), 3.25-3.15 (m, 1H), 2.49 (t, J=7.0 Hz, 2H), 2.24-2.18 (m, 2H),2.10-2.03 (m, 1H), 2.01-1.93 (m, 2H), 1.86-1.80 (m, 2H), 1.73-1.66 (m,1H); ¹³C NMR (100 MHz, CDCl₃) δ 169.9, 167.6, 163.5, 161.7, 159.7,150.7, 147.9, 139.9, 136.4, 130.2, 126.1, 125.4, 123.3, 123.0, 120.6,119.8, 114.6, 111.7 110.2, 103.9, 68.1, 56.1, 53.8, 51.2, 49.7, 39.9,37.0, 36.7, 33.0, 31.0, 29.3, 24.9, 24.5, 22.9, 18.4; MS m/z(EIMS)=681.0 (M+H)⁺; LCMS (Method A): t_(R=6.98) min.

Example 25

Allyl(6aS)-2-methoxy-3-(4-((4-(methoxycarbonyl)phenyl)-amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(25)

A solution of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (200 mg, 0.376 mmol) in anhydrous dichloromethane (5 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (150 mg, 0.394 mmol) and anhydroustriethylamine (220 μL, 1.58 mmol). The reaction mixture was stirred atroom temperature for 30 min. Methyl 4-aminobenzoate (57.0 mg, 0.376mmol) was then added and the resulting mixture was stirred at roomtemperature for 16 h. The reaction mixture was quenched with a saturatedaqueous solution of sodium hydrogen carbonate (20 mL) and extracted withdichloromethane (2×50 mL). The combined organic extracts were washedwith water containing a few drops of acetic acid (30 mL). The organiclayer was then dried over sodium sulfate, filtered and concentrated invacuo. The resulting residue was then purified by column chromatography(silica), eluting with methanol/dichloromethane (from 0% to 10%), togive the title compound (110 mg, 44%) as a yellow solid.

MS (ES+): m/z=666 (M+H)⁺; LCMS (Method A): t_(R=7.88) min.

Example 26

4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)benzoicAcid (26)

To a solution of allyl(6aS)-2-methoxy-3-(4-((4-(methoxycarbonyl)phenyl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo-[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(25) (90 mg, 0.14 mmol) in 1,4-dioxane (2.5 mL) was added an aqueoussolution of sodium hydroxide (0.5 M, 2.5 mL, 1.3 mmol). The reactionmixture was stirred at room temperature for 16 h and was thenconcentrated in vacuo, after which water (20 mL) was added and theaqueous layer was acidified to pH=1 with an aqueous solution of citricacid (1 M, 10 mL). The aqueous layer was then extracted with ethylacetate (2×50 mL). The combined organic extracts were then washed withbrine (50 mL), dried over sodium sulfate, filtered and concentrated invacuo to give the title compound (86 mg, 98%) as a cream solid. Theproduct was carried through to the next step without any furtherpurification.

MS (ES+): m/z=652 (M+H)⁺; LCMS (Method A): t_(R=7.13) min.

Example 27

Allyl(6aS)-2-methoxy-3-(4-((4-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)phenyl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-(12H)-carboxylate(27)

A solution of4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)benzoicacid (26) (40 mg, 0.061 mmol) in anhydrous dichloromethane (1 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]-pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (25 mg, 0.064 mmol) and anhydroustriethylamine (36 μL, 0.26 mmol). The reaction mixture was stirred atroom temperature for 30 min. Methyl4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (18) (14 mg, 0.061mmol) was then added and the resulting mixture was stirred at roomtemperature for 16 h. The reaction mixture was quenched with a saturatedaqueous solution of sodium hydrogen carbonate (20 mL) and extracted withdichloromethane (2×50 mL). The combined organic extracts were washedwith water containing a few drops of acetic acid (30 mL). The organiclayer was then dried over sodium sulfate, filtered and concentrated invacuo. The resulting residue was then purified by column chromatography(silica), eluting with methanol/dichloromethane (from 0% to 10%), togive the title compound (43 mg, 63%) as a yellow oil.

MS (ES+): m/z=864 (M+H)⁺; LCMS (Method A): t_(R=8.10) min.

Example 28

Methyl(S)—4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-benzamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(28)

To a solution of allyl(6aS)-2-methoxy-3-(4-((4-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)phenyl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(27) (33 mg, 0.038 mmol) in dichloromethane (3 mL) was addedtetrakis-(triphenylphosphine)palladium(0) (2.2 mg, 5 mol %),triphenylphosphine (2.5 mg, 25 mol %) and pyrrolidine (4 μL, 0.11 mmol).The reaction mixture was stirred at room temperature for 30 min. Thereaction mixture was subjected to high vacuum for 30 min until excesspyrrolidine was thoroughly removed. The resulting residue was thenpurified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 100%), to give the title compound(5.8 mg, 21%) as a yellow solid.

¹H NMR (CDCl₃, 400 MHz) δ 8.14 (br s, 1H), 8.06 (br s, 1H), 7.91 (d,J=5.7 Hz, 1H), 7.81 (d, J=8.7 Hz, 2H), 7.65 (d, J=8.6 Hz, 2H), 7.60 (d,J=8.4 Hz, 2H), 7.46-7.50 (m, 2H), 7.41 (s, 1H), 7.21 (d, J=2.1 Hz, 1H),7.08 (d, J=1.9 Hz, 1H), 6.78-6.82 (m, 1H), 4.24 (d, J=14.0 Hz, 1H),4.11-4.18 (m, 2H), 3.95-3.98 (m, 3H), 3.83-3.86 (m, 6H), 3.74-3.79 (m,2H), 3.18-3.30 (m, 2H), 2.60-2.66 (m, 2H), 2.28 (t, J=6.3 Hz, 2H), 1.97(d, J=6.3 Hz, 2H), 1.82-1.88 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 215.5,171.1, 167.5, 165.0, 163.4, 161.7, 150.3, 147.8, 141.3, 140.0, 136.2,130.8, 128.1, 125.6, 123.5, 123.1, 121.5, 120.6, 119.3, 114.7, 111.7,110.2, 67.9, 56.1, 51.2, 49.7, 39.8, 37.0, 34.3, 30.9, 25.6, 24.5, 23.0,18.4; MS (ES+): m/z=678 (M+H)⁺; LCMS (Method A): t_(R=7.05) min.

Example 29

Allyl(6aS)-2-methoxy-3-(4-((4-((2-(methoxycarbonyl)benzo-[b]thiophen-5-yl)carbamoyl)phenyl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a]-[1,4]diazepine-5(12H)-carboxylate(29)

A solution of4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)benzoicacid (26) (40 mg, 0.061 mmol) in anhydrous dichloromethane (1 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]-pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (25 mg, 0.064 mmol) and anhydroustriethylamine (36 μL, 0.26 mmol). The reaction mixture was stirred atroom temperature for 30 min. Methyl5-aminobenzo[b]-thiophene-2-carboxylate (13 mg, 0.063 mmol) was thenadded and the resulting mixture was stirred at room temperature for 16h. The reaction mixture was quenched with a saturated aqueous solutionof sodium hydrogen carbonate (20 mL) and extracted with dichloromethane(2×50 mL). The combined organic extracts were washed with watercontaining a few drops of acetic acid (30 mL). The organic layer wasthen dried over sodium sulfate, filtered and concentrated in vacuo. Theresulting residue was then purified by column chromatography (silica),eluting with methanol/dichloromethane (from 0% to 10%), to give thetitle compound (34 mg, 45%) as a brown oil.

MS (ES+): m/z=841 (M+H)⁺; LCMS (Method A): t_(R=8.15) min.

Example 30

Methyl(S)—5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-benzamido)benzo[b]thiophene-2-carboxylate(30)

To a solution of allyl(6aS)-2-methoxy-3-(4-((4-((2-(methoxycarbonyl)benzo[b]-thiophen-5-yl)carbamoyl)phenyl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(29) (23 mg, 0.028 mmol) in dichloromethane (1.5 mL) was addedtetrakis(triphenylphosphine)palladium(0) (1.6 mg, 5 mol %),triphenylphosphine (1.8 mg, 25 mol %) and pyrrolidine (3.0 μL, 0.11mmol). The reaction mixture was stirred at room temperature for 30 min.The reaction mixture was subjected to high vacuum for 30 min untilexcess pyrrolidine was thoroughly removed. The resulting residue wasthen purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 100%) followed bymethanol/dichloromethane (from 0% to 100%), to give the title compound(5.4 mg, 30%) as a pink solid.

¹H NMR (CDCl₃, 400 MHz) δ 8.48 (br s, 1H), 8.39 (d, J=1.9 Hz, 1H), 8.31(s, 1H), 7.97 (s, 1H), 7.91 (d, J=5.8 Hz, 1H), 7.77-7.84 (m, 3H), 7.65(dd, J=8.8, 2.0 Hz, 1H), 7.57 (d, J=8.6 Hz, 2H), 7.38 (s, 1H), 6.79 (s,1H), 4.24 (dt, J=13.7, 4.1 Hz, 1H), 4.09-4.17 (m, 2H), 3.95 (s, 3H),3.79-3.82 (m, 3H), 3.74-3.79 (m, 1H), 3.49 (d, J=3.9 Hz, 1H), 3.29-3.41(m, 1H), 3.17-3.28 (m, 1H), 2.58-2.64 (m, 2H), 2.26 (quin, J=6.2 Hz,2H), 2.05-2.13 (m, 1H), 1.92-2.01 (m, 1H), 1.83-1.87 (m, 1H), 1.07-1.19(m, 1H); ¹³C NMR (CDCl3, 100 MHz) δ 171.2, 167.5, 165.5, 163.4, 163.2,150.4, 147.8, 141.5, 140.0, 139.3, 138.0, 135.1, 134.4, 130.6, 128.2,123.0, 121.4, 120.9, 119.2, 116.4, 111.7, 110.1, 67.9, 56.0, 52.6, 49.7,39.8, 34.2, 30.9, 24.7, 24.5, 22.9, 18.3; MS (ES+): m/z=655 (M+H)⁺; LCMS(Method A): t_(R=7.00) min.

Example 31

Methyl4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazole-2-carboxamido)benzoate(31)

A solution of4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazole-2-carboxylic acid(100 mg, 0.415 mmol) in anhydrous dichloromethane (3 mL) was chargedwithN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (165 mg, 0.435 mmol) and anhydroustriethylamine (242 μL, 1.74 mmol). The reaction mixture was stirred atroom temperature for 30 min. Methyl 4-aminobenzoate (63 mg, 0.42 mmol)was then added and the resulting mixture was stirred at room temperaturefor 16 h. The reaction mixture was quenched with a saturated aqueoussolution of sodium hydrogen carbonate (20 mL) and extracted withdichloromethane (2×50 mL). The combined organic extracts were washedwith water containing a few drops of acetic acid (30 mL). The organiclayer was then dried over sodium sulfate, filtered and concentrated invacuo. The resulting residue was then purified by column chromatography(silica), eluting with methanol/dichloromethane (from 0% to 10%), togive the title compound (40 mg, 26%) as a cream solid.

¹H NMR (CDCl₃, 400 MHz) δ 9.16 (s, 1H), 8.01-8.07 (m, 2H), 7.69-7.75 (m,2H), 7.21 (br s, 1H), 6.84 (s, 1H), 4.07 (s, 3H), 3.92 (s, 3H), 1.53 (s,9H); MS (ES−): m/z=373 (M−H)⁻; LCMS (Method A): t_(R=7.68) min.

Example 32

Methyl 4-(4-amino-1-methyl-1H-imidazole-2-carboxamido)-benzoateHydrochloride (32)

Methyl4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazole-2-carboxamido)-benzoate(31) (40 mg, 0.11 mmol) was dissolved in hydrochloric acid (4 M in1,4-dioxane) (2 mL) and the reaction mixture was stirred at roomtemperature for 2 h. The reaction mixture was concentrated in vacuo togive the title compound (33 mg, 99%) as a brown solid. The product wascarried through to the next step without any further purification.

¹H NMR (MeOD, 400 MHz) δ 7.89-7.95 (m, 2H), 7.72-7.78 (m, 2H), 7.31 (s,1H), 4.01 (s, 3H), 3.80 (s, 3H); ¹³C NMR (MeOD, 100 MHz) δ 168.0, 143.6,132.5, 131.6, 126.9, 123.3, 120.6, 92.6, 68.1, 52.3, 36.7; MS (ES+):m/z=275 (M+H)⁺; LCMS (Method A): t_(R=5.43) min.

Example 33

Allyl(6aS)-2-methoxy-3-(4-((2-((4-(methoxycarbonyl)phenyl)-carbamoyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepine-5(12H)-carboxylate(33)

A solution of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (50 mg, 0.094 mmol) in anhydrous dichloromethane (0.5 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (38 mg, 0.099 mmol) and anhydroustriethylamine (55 μL, 0.40 mmol). The reaction mixture was stirred atroom temperature for 30 min. Methyl4-(4-amino-1-methyl-1H-imidazole-2-carboxamido)-benzoate hydrochloride(32) (30 mg, 0.094 mmol) was then added and the resulting mixture wasstirred at room temperature for 16 h. The reaction mixture was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (20 mL)and extracted with dichloromethane (2×50 mL). The combined organicextracts were washed with water containing a few drops of acetic acid(30 mL). The organic layer was then dried over sodium sulfate, filteredand concentrated in vacuo. The resulting residue was then purified bycolumn chromatography (silica), eluting with methanol/dichloromethane(from 0% to 10%), to give the title compound (72 mg, 97%) as a brownoil.

MS (ES+): m/z=789 (M+H)⁺; LCMS (Method A): t_(R=7.87) min.

Example 34

Methyl(S)—4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzoate(34)

To a solution of allyl(6aS)-2-methoxy-3-(4-((2-((4-(methoxycarbonyl)phenyl)-carbamoyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(33) (72 mg, 0.091 mmol) in dichloromethane (2 mL) was addedtetrakis(triphenylphosphine)palladium(0) (5.3 mg, 5 mol %),triphenylphosphine (6.0 mg, 25 mol %) and pyrrolidine (9.0 μL, 0.11mmol). The reaction mixture was stirred at room temperature for 30 min.The reaction mixture was subjected to high vacuum for 30 min untilexcess pyrrolidine was thoroughly removed. The resulting residue wasthen purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 100%), to give the title compound(15 mg, 27%) as a yellow solid.

¹H NMR (CDCl₃, 400 MHz) δ 9.13 (s, 1H), 8.24 (s, 1H), 8.03 (d, J=8.7 Hz,2H), 7.90 (d, J=5.7 Hz, 1H), 7.65-7.75 (m, 2H), 7.43-7.50 (m, 2H),6.77-6.83 (m, 1H), 4.12-4.23 (m, 2H), 4.07 (s, 3H), 3.93 (s, 3H), 3.91(s, 3H), 3.18-3.27 (m, 1H), 2.80 (s, 3H), 2.56-2.68 (m, 3H), 2.23-2.31(m, 2H), 1.85 (d, J=10.1 Hz, 4H); ¹³C NMR (CDCl₃, 100 MHz) δ 169.3,167.1, 166.2, 162.9, 156.2, 150.1, 147.6, 147.4, 141.4, 139.6, 135.6,132.8, 130.5, 130.3, 125.2, 121.1, 118.2, 114.8, 111.2, 109.9, 94.1,67.4, 63.5, 55.7, 53.4, 51.6, 49.2, 39.4, 38.2, 35.5, 32.5, 31.6, 30.9,28.9, 24.9, 24.3, 24.1, 22.5, 19.9, 18.0; MS (ES+): m/z=603 (M+H)⁺; LCMS(Method A): t_(R=6.57) min.

Example 35

Methyl4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzoate(35)

A solution of4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid(100 mg, 0.416 mmol) in N,N—dimethylformamide (3 mL) was charged with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (145 mg,0.756 mmol) and 4-(dimethylamino)pyridine (115 mg, 0.941 mmol). Thereaction mixture was stirred at room temperature for 3 h. Methyl4-aminobenzoate (57 mg, 0.38 mmol) was then added and the resultingmixture was stirred at room temperature for 16 h. This was then pouredonto ice-water (40 mL) and extracted with ethyl acetate (3×100 mL). Thecombined organic extracts were sequentially washed with an aqueoussolution of citric acid (1 M, 60 mL), a saturated aqueous solution ofsodium hydrogen carbonate (70 mL), water (70 mL) and brine (70 mL). Theorganic layer was then dried over sodium sulfate, filtered andconcentrated. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%), to give the title compound (90 mg, 58%) as a white solid.

¹H NMR (CDCl₃, 400 MHz) δ 7.99-8.07 (m, 2H), 7.69 (s, 1H), 7.61-7.67 (m,2H), 6.88 (s, 1H), 6.69 (br s, 1H), 6.25 (br s, 1H), 3.93 (s, 3H), 3.91(s, 3H), 1.52 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 166.6, 159.4, 153.4,142.3, 130.9, 125.5, 123.1, 122.5, 119.2, 118.7, 140.1, 80.5, 52.0,36.8, 28.4; MS (ES+): m/z=374 (M+H)⁺; LCMS (Method A): t_(R=7.52) min.

Example 36

Methyl 4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)-benzoateHydrochloride (36)

Methyl4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)-benzoate(35) (90 mg, 0.24 mmol) was dissolved in hydrochloric acid (4 M in1,4-dioxane) (3 mL) and the reaction mixture was stirred at roomtemperature for 16 h. The reaction mixture was concentrated in vacuo togive the title compound (79 mg, 99%) as a cream solid. The product wascarried through to the next step without any further purification.

¹H NMR (MeOD, 400 MHz) δ 7.99 (d, J=8.7 Hz, 2H), 7.80 (d, J=8.7 Hz, 2H),7.13 (d, J=1.9 Hz, 1H), 7.09 (d, J=1.9 Hz, 1H), 3.96 (s, 3H), 3.89 (s,3H); ¹³C NMR (MeOD, 100 MHz) δ 168.2, 161.2, 144.5, 131.5, 126.9, 126.4,123.7, 120.8, 114.2, 109.0, 52.5, 37.5; MS (ES+): m/z=274 (M+H)⁺; LCMS(Method A): t_(R=4.98) min.

Example 37

Allyl(6aS)-2-methoxy-3-(4-((5-((4-(methoxycarbonyl)phenyl)-carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepine-5(12H)-carboxylate(37)

A solution of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)-oxy)butanoicacid (9) (50 mg, 0.094 mmol) in anhydrous dichloromethane (0.5 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (38 mg, 0.099 mmol) and anhydroustriethylamine (55, L, 0.40 mmol). The reaction mixture was stirred atroom temperature for 30 min. Methyl4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)benzoate hydrochloride (36)(30 mg, 0.094 mmol) was then added and the resulting mixture was stirredat room temperature for 16 h. The reaction mixture was quenched with asaturated aqueous solution of sodium hydrogen carbonate (20 mL) andextracted with dichloromethane (2×50 mL). The combined organic extractswere washed with water containing a few drops of acetic acid (30 mL).The organic layer was then dried over sodium sulfate, filtered andconcentrated in vacuo. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%), to give the title compound (72 mg, 97%) as a brown oil.

MS (ES+): m/z=788 (M+H)⁺; LCMS (Method A): t_(R=7.77) min.

Example 38

Methyl(S)—4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)benzoate(38)

To a solution of allyl(6aS)-2-methoxy-3-(4-((5-((4-(methoxycarbonyl)phenyl)-carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(37) (72 mg, 0.091 mmol) in dichloromethane (2 mL) was addedtetrakis-(triphenylphosphine)palladium(0) (5.3 mg, 5 mol %),triphenylphosphine (6.0 mg, 25 mol %) and pyrrolidine (9.0 μL, 0.11mmol). The reaction mixture was stirred at room temperature for 30 min.The reaction mixture was subjected to high vacuum for 30 min untilexcess pyrrolidine was thoroughly removed. The resulting residue wasthen purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 100%), to give the title compound(15.0 mg, 27%) as a yellow solid.

¹H NMR (CDCl₃, 400 MHz) δ 8.41 (s, 1H), 8.00 (s, 2H), 7.98 (s, 1H), 7.90(d, J=5.8 Hz, 1H), 7.72-7.74 (m, 1H), 7.70-7.72 (m, 1H), 7.41 (s, 1H),7.14 (d, J=1.8 Hz, 1H), 6.79 (s, 1H), 6.57 (d, J=1.8 Hz, 1H), 4.22 (d,J=14.1 Hz, 1H), 4.09 (t, J=6.0 Hz, 2H), 3.89 (s, 3H), 3.88 (s, 3H), 3.83(s, 3H), 3.74-3.79 (m, 2H), 3.21 (d, J=3.3 Hz, 1H), 2.47-2.52 (m, 2H),2.17-2.23 (m, 2H), 1.93 (br s, 3H), 1.79-1.85 (m, 2H); ¹³C NMR (CDCl₃,100 MHz) δ 170.0, 167.6, 166.8, 163.6, 159.8, 150.7, 147.9, 142.9,139.9, 130.7, 124.9, 122.8, 121.6, 121.5, 120.8, 119.1, 111.8, 110.4,104.6, 68.1, 56.1, 52.0, 49.7, 39.9, 36.9, 33.0, 31.0, 25.0, 24.5, 22.9,18.3; MS (ES+): m/z=602 (M+H)⁺; LCMS (Method A): t_(R=6.52) min.

Example 39

4-(4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicAcid (39)

To a solution of allyl(6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(23) (195 mg, 0.225 mmol) in 1,4-dioxane (5 mL) was added an aqueoussolution of sodium hydroxide (0.5 M, 5 mL, 2.5 mmol). The reactionmixture was stirred at room temperature for 16 h and was thenconcentrated in vacuo, after which water (20 mL) was added and theaqueous layer was acidified to pH=1 with an aqueous solution of citricacid (1 M, 5 mL). The aqueous layer was then extracted with ethylacetate (2×50 mL). The combined organic extracts were then washed withbrine (50 mL), dried over sodium sulfate, filtered and concentrated togive the title compound (190 mg, 99%) as a cream solid. The product wascarried through to the next step without any further purification.

MS (ES+): m/z=853 (M+H)⁺; LCMS (Method B): T_(R=3.83) min.

Example 40

Allyl(6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(40)

A solution of 44-(4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (39) (320 mg, 0.375 mmol) in anhydrous dichloromethane (1.5 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (150 mg, 0.395 mmol) and anhydroustriethylamine (220 μL, 1.58 mmol). The reaction mixture was stirred atroom temperature for 30 min. Benzene-1,4-diamine (41 mg, 0.38 mmol) wasthen added and the resulting mixture was stirred at room temperature for16 h. The reaction mixture was quenched with a saturated aqueoussolution of sodium hydrogen carbonate (20 mL) and extracted withdichloromethane (2×50 mL). The combined organic extracts were washedwith water containing a few drops of acetic acid (30 mL). The organiclayer was then dried over sodium sulfate, filtered and concentrated invacuo. The resulting residue was then purified by column chromatography(silica), eluting with methanol/dichloromethane (from 0% to 10%), togive the title compound (250 mg, 71%) as a cream solid.

MS (ES+): m/z=944 (M+H)⁺; LCMS (Method B): T_(R=3.45) min.

Example 41

(S)—N—(4-aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butan-amido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(41)

To a solution of allyl(6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(40) (250 mg, 0.265 mmol) in dichloromethane (3 mL) was addedtetrakis(triphenylphosphine)palladium(0) (15 mg, 5 mol %),triphenylphosphine (17 mg, 25 mol %) and pyrrolidine (26 μL, 0.32 mmol).The reaction mixture was stirred at room temperature for 16 h. Thereaction mixture was subjected to high vacuum for 30 min until excesspyrrolidine was thoroughly removed. The resulting residue was thenpurified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 100%) followed by methanol/acetone(from 0% to 100%), to give the title compound (118 mg, 59%) as a yellowsolid.

¹H NMR (DMSO-d₆, 400 MHz) δ 9.88-9.96 (m, 1H), 9.81 (s, 2H), 9.50 (s,1H), 8.32 (br s, 2H), 8.00 (d, J=5.7 Hz, 1H), 7.67-7.73 (m, 2H), 7.48(d, J=8.6 Hz, 2H), 7.39 (d, J=1.8 Hz, 1H), 7.31-7.35 (m, 2H), 7.30 (d,J=1.6 Hz, 1H), 7.27 (s, 1H), 7.22 (d, J=1.5 Hz, 1H), 6.96 (d, J=1.6 Hz,1H), 6.80 (s, 1H), 6.51-6.55 (m, 2H), 4.09-4.17 (m, 1H), 3.99-4.05 (m,1H), 3.90-3.97 (m, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H),3.68-3.72 (m, 1H), 3.05-3.16 (m, 2H), 2.44 (t, J=7.3 Hz, 2H), 2.02-2.07(m, 2H), 1.81-1.91 (m, 1H), 1.68-1.78 (m, 2H), 1.56 (d, J=4.9 Hz, 2H);¹³C NMR (DMSO-d₆, 100 MHz) δ 168.8, 166.3, 164.7, 159.5, 159.2, 150.2,147.1, 144.7, 139.8, 137.0, 129.6, 128.2, 126.1, 124.6, 124.3, 122.0,121.8, 120.4, 120.2, 118.8, 113.7, 111.3, 109.6, 104.7, 67.7, 67.2,55.6, 51.1, 49.2, 38.5, 36.2, 36.1, 35.4, 31.8, 30.2, 24.7, 23.7, 22.5,17.7; MS (ES+): m/z=757 (M+H)⁺; LCMS (Method A): T_(R=5.80) min. HRMS(EI, m/z): calculated for C₄₂H₄₄N₈O₆ (M+1)⁺ 757.3457, observed 757.3457.

Example 42

Methyl5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylate(42)

A solution of4-((tert-butoxycarbonyl)amino)-1-methyl-1f-pyrrole-2-carboxylic acid(127 mg, 0.530 mmol) in N,N—dimethylformamide (1 mL) was charged with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (185 mg,0.960 mmol) and 4-(dimethylamino)pyridine (147 mg, 1.20 mmol). Thereaction mixture was stirred at room temperature for 4 h. Methyl5-aminobenzo[b]thiophene-2-carboxylate (100 mg, 0.480 mmol) was thenadded and the resulting mixture was stirred at room temperature for 16h. This was then poured onto ice-water (40 mL) and extracted with ethylacetate (3×100 mL). The combined organic extracts were sequentiallywashed with an aqueous solution of citric acid (1 M, 60 mL), a saturatedaqueous solution of sodium hydrogen carbonate (70 mL), water (70 mL) andbrine (70 mL). The organic layer was then dried over sodium sulfate,filtered and concentrated to give the title compound (185 mg, 90%) as acream solid. The product was carried through to the next step withoutany further purification.

MS (ES+): m/z=430 (M+H)⁺; LCMS (Method B): T_(R=4.07) min.

Example 43

Methyl5-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)-benzo[b]thiophene-2-carboxylateHydrochloride (43)

Methyl5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzo-[b]thiophene-2-carboxylate(42) (150 mg, 0.340 mmol) was dissolved in hydrochloric acid (4 M in1,4-dioxane) (1 mL) and the reaction mixture was stirred at roomtemperature for 16 h. The reaction mixture was concentrated in vacuo togive the title compound (118 mg, 95%) as a pale brown solid. The productwas carried through to the next step without any further purification.

MS (ES+): m/z=364 (M+H)⁺; LCMS (Method B): T_(R=2.78) min.

Example 44

Allyl(6aS)-2-methoxy-3-(4-((5-((2-(methoxycarbonyl)benzo-[b]thiophen-5-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-(12H)-carboxylate(44)

A solution4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (300 mg, 0.560 mmol) in N,N—dimethylformamide (3 mL) wascharged with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(238 mg, 1.23 mmol) and 4-(dimethylamino)pyridine (189 mg, 1.55 mmol).The reaction mixture was stirred at room temperature for 4 h. Methyl5-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylatehydrochloride (43) (225 mg, 0.620 mmol) was then added and the resultingmixture was stirred at room temperature for 16 h. This was then pouredonto ice-water (40 mL) and extracted with ethyl acetate (3×100 mL). Thecombined organic extracts were sequentially washed with an aqueoussolution of citric acid (1 M, 60 mL), a saturated aqueous solution ofsodium hydrogen carbonate (70 mL), water (70 mL) and brine (70 mL). Theorganic layer was then dried over sodium sulfate, filtered andconcentrated in vacuo. The resulting residue was then purified by columnchromatography (silica), eluting with acetone/dichloromethane (from 0%to 30%), to give the title compound (348 mg, 66%) as a brown solid.

MS (ES+): m/z=844 (M+H)⁺; LCMS (Method B): t_(R) 4.23 min.

Example 45

5-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylicAcid (45)

To a solution of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (44) (327 mg, 0.387 mmol) in 1,4-dioxane (5 mL) was added anaqueous solution of sodium hydroxide (0.5 M, 5 mL, 2.5 mmol). Thereaction mixture was stirred at room temperature for 3 h and was thenconcentrated in vacuo, after which water (20 mL) was added and theaqueous layer was acidified to pH=1 with an aqueous solution of citricacid (1 M, 5 mL). The aqueous layer was then extracted with ethylacetate (2×50 mL). The combined organic extracts were then washed withbrine (50 mL), dried over sodium sulfate, filtered and concentrated togive the title compound (315 mg, 99%) as a brown solid. The product wascarried through to the next step without any further purification.

MS (ES+): m/z=831 (M+H)⁺; LCMS (Method B): T_(R=3.82) min.

Example 46

Allyl(6aS)-3-(4-((5-((2-((4-aminophenyl)carbamoyl)benzo-[b]thiophen-5-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate

A solution of5-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylicacid (45) (50 mg, 0.060 mmol) in anhydrous dichloromethane (1 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (28 mg, 0.072 mmol) and anhydroustriethylamine (35 μL, 0.25 mmol). The reaction mixture was stirred atroom temperature for 30 min. Benzene-1,4-diamine (7.0 mg, 0.066 mmol)was then added and the resulting mixture was stirred at room temperaturefor 16 h. The reaction mixture was quenched with a saturated aqueoussolution of sodium hydrogen carbonate (20 mL) and extracted withdichloromethane (2×50 mL). The combined organic extracts were washedwith water containing a few drops of acetic acid (30 mL). The organiclayer was then dried over sodium sulfate, filtered and concentrated invacuo. The resulting residue was then purified by column chromatography(silica), eluting with acetone/dichloromethane (from 0% to 50%), to givethe title compound (6.8 mg, 12%) as a yellow solid.

MS (ES+): m/z=921 (M+H)⁺; LCMS (Method B): T_(R=3.48) min

Example 47

(S)—N—(2-((4-Aminophenyl)carbamoyl)benzo[b]thiophen-5-yl)-4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamide(47)

To a solution of allyl(6aS)-3-(4-((5-((2-((4-aminophenyl)carbamoyl)benzo[b]thiophen-5-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate(46) (6.8 mg, 0.0074 mmol) in dichloromethane (1 mL) was addedtetrakis(triphenylphosphine)palladium(0) (0.4 mg, 5 mol %),triphenylphosphine (0.5 mg, 25 mol %) and pyrrolidine (1 μL, 0.012mmol). The reaction mixture was stirred at room temperature for 16 h.The reaction mixture was subjected to high vacuum for 30 min untilexcess pyrrolidine was thoroughly removed. The resulting residue wasthen purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 100%) followed bymethanol/dichloromethane (from 0% to 5%), to give the title compound(1.7 mg, 31%) as a pale yellow solid.

¹H NMR (DMSO-d₆, 400 MHz) δ 10.13 (s, 1H), 9.98-10.03 (m, 1H), 9.95 (s,1H), 8.35 8.42 (m, 1H), 8.19 (s, 1H), 8.01 (d, J=5.7 Hz, 1H), 7.95 (d,J=8.9 Hz, 1H), 7.33-7.40 (m, 2H), 7.23-7.28 (m, 2H), 7.02 (s, 1H), 6.81(s, 1H), 6.57 (d, J=8.7 Hz, 2H), 5.00 (br. s., 2H), 4.10-4.14 (m, 1H),3.86 (s, 3H), 3.83 (s, 3H), 3.65-3.74 (m, 2H), 3.15-3.19 (m, 1H),3.06-3.14 (m, 1H), 2.45 (t, J=7.5 Hz, 3H), 2.11-2.13 (m, 1H), 2.00-2.08(m, 4H) 1.74 (dd, J=9.0, 5.3 Hz, 3H); MS (ES+): m/z=734 (M+H)⁺; LCMS(Method A): T_(R=5.63) min.

General Synthetic Scheme

Example 48

Ethyl 6-(4-formyl-2-methoxyphenoxy)hexanoate (48)

A mixture of vanillin (6.5 g, 42.7 mmol), ethyl 6-bromohexanoate (8.0mL, 45.0 mmol) and potassium carbonate (8.70 g, 63.0 mmol) inN,N—dimethylformaldehyde (50 mL) was stirred at room temperature for 18h. The reaction mixture was diluted with water (100 mL), separated andextracted with ethyl acetate (120 mL). The combined organic extractswere sequentially washed with water (100 mL), brine (100 mL), dried overmagnesium sulfate, filtered and concentrated to give the title compoundas a pale yellow oil (12.5 g, 99%). The product was carried through tothe next step without any further purification.

¹H NMR (400 MHz, CDCl₃) δ 9.84 (s, 1H), 7.42-7.44 (dd, J=8.2, 1.9 Hz,1H), 7.40 (d, J=1.9 Hz, 1H), 6.96 (d, J=8.1 Hz, 1H), 4.08-4.15 (m, 4H),3.92 (s, 3H), 2.34 (t, J=7.5 Hz, 2H), 1.87-1.94 (m, 2H), 1.68-1.75 (m,2H), 1.49-1.56 (m, 2H), 1.25 (t, J=7.2 Hz, 3H); MS (ES+): m/z=317(M+Na)⁺; LCMS (Method B): T_(R=3.82) min.

Example 49

Ethyl 6-(4-formyl-2-methoxy-5-nitrophenoxy)hexanoate (49)

To a stirred solution of potassium nitrate (5.4 g, 53 mmol) intrifluoroacetic acid (25 mL) at room temperature was added dropwise asolution of ethyl 6-(4-formyl-2-methoxyphenoxy)hexanoate (48) (12.5, 42mmol) in trifluoroacetic acid (25 mL). The reaction mixture was stirredfor 1 h. It was then concentrated in vacuo and the residue was dissolvedin ethyl acetate (200 mL). This was washed with brine (3×50 mL) followedby a saturated aqueous solution of sodium hydrogen carbonate (2×40 mL),dried over magnesium sulfate, filtered and concentrated in vacuo to givethe title compound as a yellow solid (14.4 g, 100%). The product wascarried through to the next step without any further purification.

¹H NMR (400 MHz, CDCl₃) δ 10.43 (s, 1H) 7.58 (s, 1H), 7.40 (s, 1H),4.10-4.16 (m, 4H), 4.00 (s, 3H), 2.35 (t, J=7.4 Hz, 2H), 1.84-1.96 (m,2H), 1.69-1.76 (m, 2H), 1.50-1.58 (m, 2H), 1.25 (t, J=7.2 Hz, 3H); MS(ES+): m/z=340 (M+H)⁺; LCMS (Method B): T_(R=4.02) min.

Example 50

4-((6-Ethoxy-6-oxohexyl)oxy)-5-methoxy-2-nitrobenzoic Acid (50)

To a solution of ethyl 6-(4-formyl-2-methoxy-5-nitrophenoxy)hexanoate(49) (7.8 g, 23.0 mmol) in acetone (200 mL) was added a hot (70° C.)solution of potassium permanganate (13.6 g, 86.0 mmol) in water (100ml). The mixture was then stirred at 70° C. for 4 h. The reactionmixture was cooled to room temperature and passed through celite. Thecake was then washed with hot water (100 mL). A solution of sodiumbisulfite in hydrochloric acid (100 mL) was added to the filtrate andextracted with dichloromethane (2×200 mL). The combined organic extractswere dried over sodium sulfate, filtrated and concentrated in vacuo togive the title compound as a yellow solid (5.0 g, 61%) which was used inthe subsequent step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.34 (s, 1H), 7.14 (s, 1H), 3.96-4.03 (m, 4H),3.84 (s, 3H), 2.24 (t, J=7.4 Hz, 2H), 1.70-1.77 (m, 2H), 1.55-1.62 (m,2H), 1.39-1.45 (m, 2H), 1.13 (t, J=7.1 Hz, 3H); MS (ES+): m/z=354(M−H)⁺; LCMS (Method B): T_(R=3.63) min.

Example 51

Ethyl(S)—6-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)hexanoate(51)

To a stirred solution of4-((6-ethoxy-6-oxohexyl)oxy)-5-methoxy-2-nitrobenzoic acid (50) (2.0 g,5.6 mmol) and trimethylamine (4.70 mL, 33.8 mmol) in dichloromethane (40mL) was added O-(7-azabenzotriazole-1-yl)-N,N,N,N,N′-tetramethyluroniumhexafluorophosphate (2.2 g, 5.9 mmol) in one portion and the resultingmixture was stirred for 2 h at room temperature. A solution of(S)—piperidin-2-ylmethanol (647 mg, 5.63 mmol) in dichloromethane (10mL) was then added dropwise and the resulting mixture was stirred for 16h at room temperature. The reaction was quenched with a saturatedaqueous solution of sodium hydrogen carbonate (40 mL), the phases wereseparated and the aqueous layer was further extracted withdichloromethane (20 mL). The combined organic extracts were washed withbrine (40 mL), dried over magnesium sulfate, filtered and concentratedto give an amber oil. Purification was carried out by columnchromatography (silica), eluting with ethyl acetate/hexane (from 0% to100%), to give the title compound (1.2 g, 48%) as a colourless oil.

¹H NMR (400 MHz, CDCl₃) δ 7.60-7.63 (m, 1H), 6.75-6.77 (m, 1H),4.02-4.13 (m, 4H), 3.93 (s, 3H), 3.70-3.78 (m, 1H), 3.39-3.68 (m, 1H),3.11-3.18 (m, 3H), 2.32 (t, J=7.6 Hz, 2H), 1.83-1.91 (m, 2H), 1.39-1.72(m, 11H), 1.26 (t, J=7.1 Hz, 3H); MS (ES+): m/z=453 (M+H)⁺; LCMS (MethodB): T_(R=3.63) min.

Example 52

Ethyl(S)—6-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)hexanoate(52)

To a solution of ethyl(S)—6-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)hexanoate(51) (1.2 g, 2.7 mmol) in methanol (20 mL) was added Raney®-Nickel(slurry in H₂O) (120 mg). The resulting mixture was hydrogenated at 50psi for 1.5 h in a Parr apparatus, then filtered through a celite padand concentrated in vacuo to give the title compound (991 mg, 87%) as agrey oil that solidifies upon standing. The resulting material wascarried through to the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 6.69 (s, 1H), 6.32 (s, 1H), 4.13 (m, 4H), 3.98(t, J=6.5 Hz, 2H), 3.79 (s, 3H), 3.67-3.57 (m, 1H), 3.19-3.22 (m, 4H),2.87 (s, 2H), 2.32-2.36 (m, 2H), 1.82-1.89 (m, 2H), 1.65-1.73 (m, 6H),1.47-1.55 (m, 3H), 1.27 (t, J=7.1 Hz, 3H); MS (ES+): m/z=423 (M+H)⁺;LCMS (Method B): T_(R=3.23) min.

Example 53

Ethyl(S)—6-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxy-methyl)piperidine-1-carbonyl)-2-methoxyphenoxy)hexanoate(53)

To a solution of ethyl(S)—6-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)hexanoate(52) (1.23 g, 2.91 mmol) and pyridine (542 μL, 6.69 mmol) in anhydrousdichloromethane (20 mL) at −10° C., a solution of allyl chloroformate(278 μL, 2.62 mmol) in dichloromethane (12 mL) was added dropwise. Theresulting reaction mixture was stirred at room temperature for 0.5 h,quenched with a saturated aqueous solution of copper (II) sulfate (25mL), diluted with dichloromethane (10 mL), separated, and successivelywashed with water (20 mL), a saturated aqueous solution of sodiumhydrogen carbonate (20 mL) and brine (20 mL). The organic layer was thendried over magnesium sulfate, filtered and concentrated in vacuo to givethe title compound (588 mg, 40%) as an orange oil. The resultingmaterial was carried through to the next step without furtherpurification.

¹H NMR (400 MHz, CDCl₃) δ 8.23 (br s, 1H), 7.70 (br s, 1H), 6.78 (s,1H), 5.90-6.00 (m, 1H), 5.33-5.38 (m, 1H), 5.24 (dd, J=10.4, 1.3 Hz,1H), 4.63 (m, 2H), 4.12 (q, J=7.1 Hz, 2H) 4.05 (t, J=6.6 Hz, 2H), 3.83(s, 3H), 3.64-3.72 (m, 1H), 3.02-3.12 (m, 1H), 2.33 (t, J=7.6 Hz, 2H),1.84-1.91 (m, 2H), 1.67-1.74 (m, 10H), 1.66-1.54 (m, 4H), 1.26 (t, J=7.1Hz, 3H); MS (ES+): m/z=507 (M+H)⁺; LCMS (Method B): T_(R=3.70) min.

Example 54

Allyl(6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate(54)

To a solution of ethyl(S)—6-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)-piperidine-1-carbonyl)-2-methoxyphenoxy)hexanoate(53) (1.7 g, 3.4 mmol) in dichloromethane (80 mL) was added2,2,6,6-tetramethyl-1-piperidinyloxy (53 mg, 0.30 mmol) and(diacetoxyiodo)benzene (1.3 g, 4.0 mmol). The reaction mixture wasstirred at room temperature for 16 h, and was then placed in an ice bathand quenched with a saturated aqueous solution of sodium metabisulfite(35 mL). The mixture was diluted with dichloromethane (30 mL),separated, and sequentially washed with a saturated aqueous solution ofsodium hydrogen carbonate (30 mL), water (30 mL) and brine (30 mL). Theorganic layer was then dried over magnesium sulfate, filtered andconcentrated in vacuo. Purification was carried out by columnchromatography (silica), eluting with ethyl acetate/hexane (from 0% to80%) to give the desired compound (1.1 g, 66%) as a colourless oil.

¹H NMR (400 MHz, CDCl₃) δ 7.70-7.72 (m, 1H), 7.09-7.13 (m, 1H),5.80-5.98 (m, 1H), 5.25-5.38 (m, 1H), 5.14-5.19 (m, 2H), 4.63-4.72 (m,2H), 4.35-4.50 (m, 1H), 4.13 (q, J=7.1 Hz, 2H), 4.03-4.08 (m, 1H),3.96-4.01 (m, 2H), 3.91 (s, 3H), 3.81-3.83 (m, 1H), 3.45-3.53 (m, 1H),3.03-3.10 (m, 1H), 2.33 (t, J=7.6 Hz, 2H), 1.83-1.90 (m, 2H), 1.62-1.74(m, 10H), 1.48-1.53 (m, 2H); MS (ES+): m/z=505 (M+H)⁺; LCMS (Method B):T_(R=3.57) min.

Example 55

Allyl(6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido-[1,2-a][1,4]diazepine-5(12H)-carboxylate(55)

To containing solution of allyl(6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(54) (1.1 g, 2.2 mmol) in dichloromethane (50 mL) were added3,4-dihydro-2H-pyran (2.00 mL, 22.4 mmol) and p-toluenesulfonic acidmonohydrate (113 mg, 1% w/w), and the resulting mixture was stirred atroom temperature for 4 h. The reaction mixture was then diluted withdichloromethane (50 mL) and washed with a saturated aqueous solution ofsodium hydrogen carbonate (50 mL) and brine (50 mL). The organic layerwas dried over magnesium sulfate, filtered and concentrated to give thetitle compound as a yellow oil (863 mg, 66%) after purification bycolumn chromatography (silica) eluting with ethyl acetate/hexane (from0% to 70%).

¹H NMR (400 MHz, CDCl₃) δ 7.16 (m, 1H), 6.50 (s, 1H), 6.10 (m, 1H),5.76-5.81 (m, 1H), 5.03-5.14 (m, 2H), 4.57-4.69 (m, 2H), 4.37-4.49 (m,1H), 4.26-4.34 (m, 1H), 4.12 (q, J=7.1 Hz, 2H), 3.94-4.01 (m, 3H), 3.90(s, 3H), 3.62-3.68 (m, 1H), 3.46-3.68 (m, 2H), 3.03-3.12 (m, 1H), 2.33(t, J=7.4 Hz, 2H), 1.66-1.89 (m, 11H), 1.47-1.57 (m, 6H), 1.25 (t, J=7.1Hz, 3H); MS (ES+): m/z=589 (M+H)⁺; LCMS (Method B): T_(R=4.32) min.

Example 56

6-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoicAcid (56)

To a solution of allyl(6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate(55) (200 mg, 0.34 mmol) in 1,4-dioxane (3 ml) was added an aqueoussolution of sodium hydroxide (0.5 M, 1.2 mL). The reaction mixture wasstirred at room temperature for 2 h and was then concentrated in vacuo,after which water (6 ml) was added and the aqueous layer was thenacidified to pH=1 with acetic acid. The aqueous layer was then extractedwith ethyl acetate (2×40 mL). The combined organic extracts were thenwashed with brine (40 ml), dried over sodium sulfate, filtered andconcentrated to give the title compound as a yellow oil (181 mg, 95%)which was used in the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.18 (s, 1H), 6.19 (s, 1H), 5.99-6.19 (m, 1H),5.71-5.81 (m, 1H), 5.02-5.12 (m, 2H), 4.51-4.67 (m, 1H), 4.36-4.48 (m,1H), 4.23-4.31 (m, 1H), 3.88-4.00 (m, 7H), 3.46-3.66 (m, 2H), 3.02-3.12(m, 1H), 2.36 (t, J=7.4 Hz, 2H), 1.79-1.81 (m, 2H), 1.65-1.75 (m, 10H),1.49-1.55 (m, 7H); MS (ES+): m/z=561 (M+H)⁺; LCMS (Method B): T_(R=3.78)min.

Example 57

Methyl 4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2carboxamido)phenyl)-1-methyl-1H-pyrrole-2 carboxylate (57)

To a solution of4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid(18) (59 mg, 0.23 mmol) in N,N—dimethylformamide (4 mL) was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (67 mg, 0.36mmol) and 4-(dimethylamino)pyridine (65 mg, 0.53 mmol). The reactionmixture was stirred at room temperature for 2 h. Methyl4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (41 mg, 0.18 mmol)was added to the reaction mixture which was then stirred at roomtemperature for 16 h. The reaction mixture was poured into ice-water (40mL) and extracted with ethyl acetate (3×100 mL). The combined organiclayer was sequentially washed with 1 M citric acid (60 mL), a saturatedaqueous solution of sodium hydrogen carbonate (70 mL), water (70 mL) andbrine (70 mL). The organic layer was dried over sodium sulfate, filteredand concentrated. The resulting residue was purified by columnchromatography (silica), eluting with ethyl acetate/dichloromethane(from 0% to 50%), to give the title compound (36 mg, 45%) as a creamsolid.

MS (ES+): m/z=453 (M+H)⁺; LCMS (Method B): T_(R=4.07) min.

Example 58

Methyl4-(4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)-phenyl)-1-methyl-1H-pyrrole-2-carboxylate(58)

Methyl4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)-phenyl)-1-methyl-1H-pyrrole-2-carboxylate(57) (150 mg, 0.330 mmol) was dissolved in hydrochloric acid (4 M in1,4-dioxane) (1 mL) and the reaction mixture was stirred at roomtemperature for 2 h. The reaction mixture was concentrated in vacuo togive the title compound (114 mg, 99%) as a brown solid. The product wascarried through to the next step without further purification.

MS (ES+): m/z=353 (M+H)⁺; LCMS (Method B): T_(R=2.88) min.

Example 59

Allyl(6aS)-2-methoxy-3-((6-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(59)

A solution of6-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoicacid (56) (194 mg, 0.360 mmol) in N,N—dimethylformamide (5 mL) wascharged with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(126 mg, 0.660 mmol) and 4-(dimethylamino)pyridine (121 mg, 0.990 mmol).The reaction mixture was stirred at room temperature for 3 h. Methyl4-(4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(58) (150 mg, 0.330 mmol) was then added and the resulting mixture wasstirred at room temperature for 16 h. This was then poured ontoice-water (20 mL) and extracted with ethyl acetate (3×75 mL). Thecombined organic extracts were sequentially washed with an aqueoussolution of citric acid (1 M, 50 mL), a saturated aqueous solution ofsodium hydrogen carbonate (50 mL), water (50 mL) and brine (50 mL). Theorganic layer was then dried over sodium sulfate, filtered andconcentrated in vacuo to give the title compound (133 mg, 45%) as ayellow oil. The product was carried through to the next step withoutfurther purification.

MS (ES+): m/z=896 (M+H)⁺; LCMS (Method B): T_(R=4.25) min.

Example 60

4-(4-(4-(6-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicAcid (60)

To a solution of allyl(6aS)-2-methoxy-3-((6-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(59) (200 mg, 0.340 mmol) in 1,4-dioxane (3 ml) was added an aqueoussolution of sodium hydroxide (1 M, 1.2 mL). The reaction mixture wasstirred at room temperature for 2 h and was then concentrated in vacuo,after which water (6 ml) was added and the aqueous layer was acidifiedto pH=1 with acetic acid. The aqueous layer was then extracted withethyl acetate (2×40 mL). The combined organic extracts were then washedwith brine (40 ml), dried over sodium sulfate, filtered and concentratedto give the title compound as a yellow oil (181 mg, 95%) which was usedin the next step without further purification.

MS (ES+): m/z=882 (M+H)⁺; LCMS (Method B): T_(R=3.92) min.

Example 61

Allyl(6aS)-3-((6-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(61)

A solution of4-(4-(4-(6-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (60) (123 mg, 0.14 mmol) in anhydrous dichloromethane (2 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (54 mg, 0.14 mmol) and anhydroustriethylamine (117 μL, 0.84 mmol). The reaction mixture was stirred atroom temperature for 30 min. Benzene-1,4-diamine (15.1 mg, 0.14 mmol)was then added and the resulting mixture was stirred at room temperaturefor 16 h. The reaction mixture was quenched with a saturated aqueoussolution of sodium hydrogen carbonate (20 mL) and extracted withdichloromethane (2×50 mL). The combined organic extracts were washedwith water containing a few drops of acetic acid (30 mL). The organiclayer was then dried over sodium sulfate, filtered and concentrated invacuo. The resulting residue was then purified by column chromatography(silica), eluting with acetone/dichloromethane (from 0% to 50%), to givethe title compound (63 mg, 46%) as a yellow solid.

MS (ES+): m/z=972 (M+H)⁺; LCMS (Method B): T_(R=3.55) min

Example 62

(S)—N—(4-aminophenyl)-4-(4-(4-(4((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydroxybenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexan-amido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(62)

To a solution of Allyl(6aS)-3-((6-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(61) (25 mg, 0.026 mmol) in dichloromethane (1 mL) was addedtetrakis(triphenylphosphine)palladium(0) (2.5 mg, 5 mol %),triphenylphosphine (1.7 mg, 25 mol %) and pyrrolidine (21 μL, 0.260mmol). The reaction mixture was stirred at room temperature for 16 h.The reaction mixture was subjected to high vacuum for 30 min untilexcess pyrrolidine was thoroughly removed. The resulting residue wasthen purified by column chromatography (silica), eluting withacetate/hexane (from 0% to 100%) to give the title compound (6.8 mg,33%) as a pale yellow solid.

¹H NMR (DMSO-d₆, 400 MHz) δ 9.81-9.85 (m, 1H), 9.58 (s, 1H), 9.51 (s,2H), 8.00 (d, J=5.7 Hz, 1H), 7.69-7.72 (m, 2H), 7.47-7.49 (m, 2H),7.38-7.43 (m, 1H), 7.30-7.35 (m, 2H), 7.18-7.24 (m, 1H), 7.11-7.13 (m,1H), 7.07 (s, 1H), 6.94-6.98 (m, 1H), 6.80 (br s, 1H), 6.63-6.72 (m,2H), 6.52-6.54 (m, 1H), 3.95-4.14 (m, 3H), 3.89 (s, 3H), 3.83 (s, 3H),3.70 (s, 3H), 3.65-3.69 (m, 1H), 3.17 (d, J=5.2 Hz, 2H), 2.28 (t, J=6.5Hz, 2H), 1.72-1.78 (m, 4H), 1.62-1.68 (m, 4H), 1.42-1.48 (m, 3H) ¹³C NMR(DMSO-d₆, 100 MHz) δ 169.5, 166.3, 164.6, 159.5, 159.2, 150.3, 147.1,144.8, 139.8, 137.0, 129.6, 128.2, 126.6, 124.6, 124.3, 122.7, 122.1,121.8, 121.7, 120.5, 120.4, 118.7, 113.7, 111.3, 109.6, 109.3, 104.7,68.1, 55.6, 36.3, 36.1, 35.5, 28.3, 25.2, 25.1, 23.7, 22.5, 17.7; MS(ES+): m/z=785 (M+H)⁺; LCMS (Method A): T_(R=3.08) min.

Example 63

Allyl(6S,6aS)-2-methoxy-3-((6-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]-diazepine-5(12H)-carboxylate(63)

A solution of6-(((6S,6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoicacid (56) (109 mg, 0.190 mmol) in anhydrous dichloromethane (3 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (76 mg, 0.20 mmol) and anhydroustriethylamine (115 μL, 1.14 mmol). The reaction mixture was stirred atroom temperature for 30 min. Methyl4-amino-1-methyl-1H-pyrrole-2-carboxylate (37 mg, 0.24 mmol) was thenadded and the resulting mixture was stirred at room temperature for 16h. The reaction mixture was quenched with a saturated aqueous solutionof sodium hydrogen carbonate (20 mL) and extracted with dichloromethane(2×50 mL). The combined organic extracts were washed with watercontaining a few drops of acetic acid (30 mL). The organic layer wasthen dried over sodium sulfate, filtered and concentrated in vacuo. Theresulting residue was then purified by column chromatography (silica),eluting with acetone/dichloromethane (from 0% to 30%), to give the titlecompound (82 mg, 62%) as a white solid.

MS (ES+): m/z=697 (M+H)⁺; LCMS (Method B): T_(R=3.98) min.

Example 64

4-(6-(((6S,6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]-pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanamido)-1-methyl-1H-pyrrole-2-carboxylicAcid (64)

To a solution of allyl(6S,6aS)-2-methoxy-3-((6-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(63) (76 mg, 0.11 mmol) in 1,4-dioxane (1 mL) was added an aqueoussolution of sodium hydroxide (0.5 M, 1.0 mL, 0.50 mmol). The reactionmixture was stirred at room temperature for 16 h and was thenconcentrated in vacuo, after which water (20 mL) was added and theaqueous layer was acidified to pH=1 with an aqueous solution of citricacid (1 M, 10 mL). The aqueous layer was then extracted with ethylacetate (2×50 mL). The combined organic extracts were then washed withbrine (50 mL), dried over sodium sulfate, filtered and concentrated invacuo to give the title compound (74 mg, 98%) as a cream solid. Theproduct was carried through to the next step without any furtherpurification.

MS (ES+): m/z=683 (M+H)⁺; LCMS (Method B): T_(R=3.68) min.

Example 65

Allyl(6S,6aS)-3-((6-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(65)

A solution of4-(6-(((6S,6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanamido)-1-methyl-1H-pyrrole-2-carboxylicacid (64) (60 mg, 0.090 mmol) in anhydrous dichloromethane (1 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (67.0 mg, 0.175 mmol) and anhydroustriethylamine (73 μL, 0.52 mmol). The reaction mixture was stirred atroom temperature for 30 min. Benzene-1,4-diamine (10 mg, 0.10 mmol) wasthen added and the resulting mixture was stirred at room temperature for16 h. The reaction mixture was quenched with a saturated aqueoussolution of sodium hydrogen carbonate (20 mL) and extracted withdichloromethane (2×50 mL). The combined organic extracts were thenwashed with water containing a few drops of acetic acid (30 mL). Theorganic layer was then dried over sodium sulfate, filtered andconcentrated in vacuo. The resulting residue was then purified by columnchromatography (silica), eluting with acetone/dichloromethane (from 30%to 50%+5% MeOH), to give the title compound (18 mg, 26%) as a brownsolid.

MS (ES+): m/z=773 (M+H)⁺; LCMS (Method B): T_(R=3.27) min.

Example 66

(S)—N—(4-Aminophenyl)-4-(6-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexan-amido)-1-methyl-1H-pyrrole-2-carboxamide(66)

To a solution of allyl(6S,6aS)-3-((6-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(65) (18 mg, 0.020 mmol) in dichloromethane (1 mL) was addedtetrakis(triphenylphosphine)palladium(0) (1.3 mg, 5 mol %) andpyrrolidine (2.3 μL, 0.030 mmol). The reaction mixture was stirred atroom temperature for 30 min and then subjected to high vacuum for 30 minuntil excess pyrrolidine was thoroughly removed. The resulting residuewas then purified by column chromatography (silica), eluting withmethanol/dichloromethane (from 0% to 100%), to give the title compound(11.6 mg, 86%) as an off-white solid.

¹H NMR (DMSO-d₆, 400 MHz) δ 9.78 (s, 1H), 9.48 (s, 1H), 8.00 (d, J=5.7Hz, 1H), 7.32 (d, J=8.8 Hz, 2H), 7.25 (s, 1H), 7.17 (d, J=1.8 Hz, 1H),6.82 (d, J=1.9 Hz, 1H), 6.79 (s, 1H), 6.56 (d, J=8.6 Hz, 2H), 4.13 (dd,J=5.7, 3.4 Hz, 5H), 3.80 (s, 3H), 3.79 (s, 3H), 3.17 (s, 1H), 3.07-3.11(m, 1H), 2.26 (t, J=7.2 Hz, 3H), 1.75 (dd, J=13.8, 7.0 Hz, 6H),1.60-1.65 (m, 5H); MS (ES+): m/z=587 (M+H)⁺; LCMS (Method B): T_(R=2.72)min, MS (ES+): m/z=587 (M+H)⁺; LCMS (Method A): T_(R=5.23) min.

Example 67

Allyl(6aS)-3-(4-((5-((4-(5-((2-aminoethyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(67)

A solution of4-(4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]-diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (39) (270 mg, 0.317 mmol) in anhydrous dichloromethane (6 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (126 mg, 0.333 mmol) and anhydroustriethylamine (185 μL, 1.33 mmol). The reaction mixture was stirred atroom temperature for 30 min. Ethane-1,2-diamine (379 mg, 6.33 mmol) wasthen added and the resulting mixture was stirred at room temperature for16 h. The reaction mixture was quenched with a saturated aqueoussolution of sodium hydrogen carbonate (20 mL) and extracted withdichloromethane (2×50 mL). The combined organic extracts were washedwith water containing a few drops of acetic acid (30 mL). The organiclayer was then dried over sodium sulfate, filtered and concentrated invacuo. The resulting residue was then purified by column chromatography(silica), eluting with ammonia in methanol (2 M)/dichloromethane (from0% to 10%), to give the title compound (180 mg, 63%) as a white solid.

MS (ES+): m/z=896 (M+H)⁺; LCMS (Method B): T_(R=3.12) min.

Example 68

(S)—N—(2-Aminoethyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(68)

To a solution of allyl(6aS)-3-(4-((5-((4-(5-((2-aminoethyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]-pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(67) (22 mg, 0.025 mmol) in dichloromethane (4 mL) was addedtetrakis(triphenylphosphine)palladium(0) (1.4 mg, 5 mol %) andpyrrolidine (3.0 μL, 0.037 mmol). The reaction mixture was stirred atroom temperature for 2 h and then subjected to high vacuum for 30 minuntil excess pyrrolidine was thoroughly removed. The resulting residuewas then purified by column chromatography (silica), eluting withmethanol/dichloromethane (from 0% to 20%), to give the title compound(11 mg, 62%) as a white solid.

¹H NMR (DMSO-d₆, 400 MHz) δ 10.01 (s, 1H), 9.84 (s, 1H), 9.21 (br s,2H), 8.41 (s, 1H), 8.01 (d, J=5.7 Hz, 1H), 7.70 (d, J=8.8 Hz, 2H), 7.44(d, J=8.7 Hz, 2H), 7.38 (d, J=1.8 Hz, 1H), 7.31 (d, J=1.9 Hz, 1H), 7.27(s, 1H), 7.22 (d, J=1.8 Hz, 1H), 6.98 (d, J=1.8 Hz, 1H), 6.80 (s, 1H),4.09-4.19 (m, 2H), 3.99-4.05 (m, 2H), 3.87 (s, 3H), 3.82 (m, 6H),3.65-3.72 (m, 2H), 3.45-3.50 (m, 2H), 3.16 (d, J=5.3 Hz, 3H), 2.96 (t,J=5.8 Hz, 2H), 2.45 (t, J=7.4 Hz, 2H), 2.00-2.09 (m, 4H); (DMSO-d₆, 100MHz) δ 203.1, 168.8, 166.3, 164.7, 161.6, 159.6, 150.2, 147.1, 139.8,137.0, 129.5, 125.9, 124.2, 122.0, 120.6, 120.4, 111.2, 109.8, 109.3,98.8, 95.4, 85.9, 78.8, 71.0, 67.7, 55.6, 49.2, 48.5, 36.3, 31.8, 30.2,24.7, 22.5, 17.7; MS (ES+): m/z=709 (M+H)⁺; LCMS (Method B): T_(R=2.80)min, MS (ES+): m/z=709 (M+H)⁺; LCMS (Method A): T_(R=5.38) min.

Example 69

Allyl(6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(7)

To a solution of methyl(S)—4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate(6) (930 mg, 2.00 mmol) in dichloromethane (45 mL) was added2,2,6,6-tetramethyl-1-piperidinyloxy (32 mg, 0.20 mmol) and(diacetoxyiodo)benzene (773 mg, 2.40 mmol). The reaction mixture wasstirred at room temperature for 16 h, and was then sequentially washedwith a saturated aqueous solution of sodium metabisulfite (20 mL), asaturated aqueous solution of sodium hydrogen carbonate (20 mL), water(20 mL) and brine (20 mL). The organic layer was then dried over sodiumsulfate, filtered and concentrated. The resulting residue was purifiedby column chromatography (silica), eluting with methanol/dichloromethane(from 0% to 5%), to give the title compound (825 mg, 89%) as a creamsolid, mixture of diastereomers.

¹H NMR (400 MHz, CDCl₃) δ 7.12, (S, 1H), 6.63 (s, 1H), 5.87 (d, J=10.1Hz, 1H), 5.81-5.65 (m, 1H), 5.08 (d, J=12.1 Hz, 2H), 4.62 (dd, J=13.3Hz, 5.3 Hz, 1H), 4.41 (br. s., 1H), 4.31-4.21 (m, 1H), 4.08-3.95 (m,3H), 3.84 (s, 3H), 3.62 (s, 3H), 3.45-3.38 (m, 1H), 3.01 (ddd, J=3.9,10.3, 14.0 Hz, 1H), 2.48 (t, J=7.2 Hz, 3H), 2.13-2.05 (m, 3H), 1.77-1.57(m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 173.4, 169.0, 150.0, 148.9, 131.8,125.2, 117.9, 113.5, 117.9, 113.5, 110.6, 82.3, 67.9, 66.7, 56.0, 55.4,51.6, 38.6, 30.6, 30.3, 30.3, 24.2, 23.1, 22.9, 18.1; MS (ES+): m/z=463(M+H)⁺; LCMS (Method A): T_(R=6.30) min.

Example 70

Allyl(6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(8)

A mixture of allyl(6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(7) (825 mg, 1.80 mmol), 3,4-dihydro-2H-pyran (1.70 mL, 18.2 mmol) andp-toluenesulfonic acid monohydrate (8.5 mg, 1% w/w) in ethyl acetate (12mL) was stirred at room temperature for 16 h. The reaction mixture wasthen diluted with ethyl acetate (50 mL) and washed with a saturatedaqueous solution of sodium hydrogen carbonate (20 mL) and brine (30 mL).The organic layer was dried over sodium sulfate, filtered andconcentrated. The resulting residue was purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 2%), to give the title compound (820 mg, 84%) as a cream solid,mixture of diastereomers.

¹H NMR (400 MHz, CDCl₃) δ 7.50 (s, 0.6H), 7.02 (s, 0.4H), 6.74 (s,0.4H), 6.48 (s, 0.6H), 6.07 (d, J=9.8 Hz, 0.6H), 5.9 (d, J=10.2 Hz,0.4H), 5.70-5.62 (m, 1H), 5.01-4.92 (m, 3H), 4.55-4.20 (m, 2H),4.18-4.13 (m, 1H), 3.96-3.91 (m, 3H), 3.78 (s, 3H), 3.55 (s, 3H),3.40-3.34 (m, 2H), 3.00-2.91 (m, 1H), 2.24 (t, J=7.0 Hz, 2H), 2.05-2.02(m, 2H), 1.67-1.43 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 170.8,169.2, 169.0, 149.3, 132.1, 131.9, 126.4, 126.0, 116.8, 114.4, 114.0,110.6, 110.2, 100.0, 952.2, 87.9, 84.0, 67.8, 67.6, 66.3, 66.1, 63.8,60.2, 55.9, 55.3, 51.4, 38.7, 30.9, 30.6, 30.2, 30.1, 25.2, 24.1, 23.1,20.9, 20.8, 19.9, 19.6, 18.3, 18.1, 14.1 MS (ES+): m/z=547 (M+H)⁺; LCMS(Method A): T_(R=7.70) min.

Example 71

4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicAcid (9)

To a solution of allyl(6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(8) (770 mg, 1.40 mmol) in 1,4-dioxane (10 mL) was added a 0.5 M aqueoussolution of sodium hydroxide (10.0 mL, 5.00 mmol). The reaction mixturewas stirred at room temperature for 2 h and was then concentrated invacuo, after which water (20 mL) was added and the aqueous layer wasacidified to pH=4 with an aqueous solution of acetic acid (5 M, 10 mL).The aqueous layer was extracted with ethyl acetate (2×50 mL). Thecombined organic extracts were washed with brine (50 mL), dried oversodium sulfate, filtered and concentrated to give the title compound(700 mg, 93%) as a yellow oil, mixture of diastereomers. The product wascarried through to the next step without any further purification(mixture of diastereomers).

¹H NMR (400 MHz, (CD₃)₂SO) δ 12.15 (br. s., 1H), 7.03 (s, 0.6H), 7.01(s, 0.4H), 6.86 (s, 0.6H), 6.78 (s, 0.4H), 6.01 (d, J=10.1, 0.6H), 5.92(d, J=9.8, 0.4H), 5.83-5.69 (m, 1H), 5.11-4.96 (m, 3H), 4.64-4.36 (m,2H), 4.16-4.02 (m, 1H), 400-3.92 (m, 2H), 3.80 (s, 3H), 3.79-3.70 (m,2H), 3.54-3.46 (m, 1H), 2.89-2.83 (m, 1H), 2.36 (t, J=7.2 Hz, 2H),1.96-1.89 (m, 2H), 1.71-1.41 (m, 12H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ174.5, 174.4, 168.5, 168.5, 150.1, 149.1, 133.1, 127.6, 126.3, 114.5,110.7, 109.1, 99.7, 84.4, 68.0, 67.9, 56.2, 52.9, 38.5, 30.6, 30.3,30.02, 25.4, 25.3, 23.1, 23.0, 18.3; MS (ES+): m/z=533 (M+H)⁺; LCMS(Method A): T_(R=6.98) min.

Example 72

Methyl4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(70)

To a solution of methyl 4-bromo-1-methyl-1H-pyrrole-2-carboxylate (1.0g, 4.60 mmol) in acetonitrile (40 mL) and water (36 mL) tert-butyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate (1.8mg, 5.06 mmol), potassium carbonate (69) (1.7 g, 13.36 mmol), andtetrakis(triphenylphosphine)palladium (280 mg, mol 5%) were added. Thereaction mixture was purged with nitrogen for 5 min and the reaction wascarried out in a microwave reactor at 100° C. for 6 min. The mixture wasfiltered through a celite pad. The pad was washed with ethyl acetate(500 mL) and the resulting organic solution was concentrated in vacuo.The residue was purified by column chromatography (silica), eluting withethyl acetate/hexane (from 0% to 40%), to give the title compound (958mg, 63%) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 7.37-7.42 (m, 2H), 7.32-7.36 (m, 2H), 7.16 (d,J=2.0 Hz, 1H), 7.02 (d, J=2.0 Hz, 1H), 6.56 (s, 1H), 3.94 (s, 3H), 3.83(s, 3H), 1.52 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 136.5, 129.4,127.1, 125.9, 125.5, 123.6, 119.0, 115.6, 114.6, 60.4, 51.1, 36.9, 28.3;MS (ES+): m/z=330.9 (M+H)⁺; LCMS (Method B): T_(R=4.22) min.

Example 73

Methyl4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(57)

To a solution of methyl4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(70) (950 mg, 2.88 mmol) in dioxane and methanol (1:1) (8 mL)hydrochloric acid (4 M in 1,4-dioxane) (8 mL) was added drop wise. Thereaction mixture was stirred for 3 h and then concentrated in vacuo. Theresidue was added to a mixture of4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid(830 mg, 3.45 mmol), N,N—dimethylpyridin-4-amine (1.05 g, 8.64 mmol) andN—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.38 g,7.20 mmol) in N,N—dimethylformamide (15 mL) which was previously stirredfor 30 min. The resulting solution was allowed to react at roomtemperature for 18 h. The reaction mixture was quenched with a saturatedaqueous solution of sodium hydrogen carbonate (20 mL) and loaded withbrine (150 mL). The aqueous phase was extracted with ethyl acetate (2×60mL). The combined organic extracts were concentrated in vacuo. Theresulting residue was purified by column chromatography (silica),eluting with acetone/dichloromethane (from 0% to 30%), to give the titlecompound (860 mg, 66%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 8.01 (2, 1H), 7.71 (s, 1H), 7.49-7.54 (m, 2H),7.40-7.44 (m, 2H), 7.17 (d, J=2.0, 1H), 7.03 (d, J=1.8, 1H), 6.85 (s,1H), 6.63 (s, 1H), 3.94 (s, 3H), 3.88 (s, 3H), 3.83 (s, 3H) 1.50 (s,9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 159.5, 136.0, 130.4, 126.0,125.5, 123.5, 123.5, 121.8, 120.3, 118.6, 114.6, 110.0, 103.7, 51.1,36.9, 36.7, 28.4; MS (ES+): m/z=453.1 (M+H)⁺; LCMS (Method B):T_(R=4.07) min.

Example 74

Allyl(6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(23)

To a solution of methyl4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(57) (440.0 mg, 0.97 mmol) in dioxane and methanol (1:1) (4 mL)hydrochloric acid (4 M in 1,4-dioxane) (4 mL) was added drop wise. Thereaction mixture was stirred for 4 h and then concentrated in vacuo. Theresidue was added to a mixture of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (470.0 mg, 0.88 mmol), N,N—dimethylpyridin-4-amine (322.0 mg,2.64 mmol) and N—(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (421.7 mg, 2.20 mmol) in N,N—dimethylformamide (7 mL)which was previously stirred for 30 min. The resulting solution wasallowed to react at room temperature for 18 h. The reaction mixture wasquenched with a saturated aqueous solution of sodium hydrogen carbonate(10 mL) and loaded with brine (90 mL). The aqueous phase was extractedwith ethyl acetate (2×50 mL). The combined organic extracts wereconcentrated in vacuo. The resulting residue was purified by columnchromatography (silica), eluting with acetone/dichloromethane (from 0%to 30%), to give the title compound (600 mg, 78%) as an orange solid(mixture of diastereomers).

¹H NMR (400 MHz, CDCl₃) δ 7.42 (d, J=8.3 Hz, 2H), 7.22-7.10 (m, 4H),7.04 (s, 2H), 6.76 (br. s., 1H), 6.02-5.87 (m, 1H), 5.74-5.68 (m, 1H),5.38-5.25 (m, 1H), 5.11-5.05 (m, 1H), 4.38-4.26 (m, 1H), 4.11 (br. s.,2H), 3.93 (s, 3H), 3.88 (br. s., 5H), 3.82 (s, 6H), 3.78 (br. s., 2H),3.62 (br. s., 3H), 2.48-2.39 (m, 2H), 2.12-2.03 (m, 2H), 1.75-1.50 (m,12H); ¹³C NMR (100 MHz, CDCl₃) δ 184.1, 169.7, 169.7, 169.6, 169.2,168.0, 161.6, 159.9, 136.5, 130.0, 127.8, 127.6, 126.0, 125.2, 123.5,122.9, 122.8, 121.8, 121.6, 121.6, 120.7, 120.6, 117.6, 114.6, 104.0,56.0, 55.9, 51.1, 51.1, 36.9, 36.7, 30.9, 30.7, 25.1, 23.2; MS (ES+):m/z=867.4 (M+H)⁺; LCMS (Method B): T_(R=4.17) min.

Example 75

4-(4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicAcid (71)

To a solution of allyl(6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(23) (600 mg, 0.69 mmol) in 1,4-dioxane (10 mL) was added an aqueoussolution of sodium hydroxide (1 M, 10 mL, 10 mmol). The reaction mixturewas stirred at room temperature for 18 h and was then concentrated invacuo, after which water (100 mL) was added and the aqueous layer wasacidified to pH=4 with an aqueous solution of acetic acid (5 M, 20 mL).The aqueous layer was then extracted with ethyl acetate (2×100 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated to give the title compound (558 mg, 97%) as a cream solid.The product was carried through to the next step without any furtherpurification (mixture of diastereomers).

¹H NMR (400 MHz, CD₃OD) δ 7.58-7.54 (m, 2H), 7.46 (d, J=8.3 Hz, 2H),7.24 (s, 1H), 7.18 (s, 2H), 7.13 (s, 1H), 6.88 (br. s., 2H), 6.17 (d,J=9.8 Hz, 1H), 5.78-5.74 (m, 1H), 4.66-4.38 (m, 3H), 4.26-4.12 (m, 1H),4.06 (m, 3H), 3.91 (s, 3H), 3.87 (s, 3H), 3.84 (br. s., 4H), 3.67-3.49(m, 2H), 3.44 (br. s., 1H), 3.11-2.96 (m, 1H), 2.51 (t, J=7.30 Hz, 2H),2.15-2.12 (m, 2H), 1.72-1.48 (m, 12H); ¹³C NMR (100 MHz, CD₃OD) δ 175.6,172.2, 171.4, 164.6, 162.2, 152.1, 150.9, 137.8, 133.5, 132.1, 129.2,127.6, 126.1, 125.0, 124.7, 124.6, 123.4, 122.4, 117.6, 115.8, 115.6,106.4, 85.5, 69.5, 67.7, 56.6, 40.2, 37.3, 37.0, 31.8, 26.5, 26.4, 24.0,21.0, 20.6, 19.1; MS (ES+): m/z=853 (M+H)⁺; LCMS (Method B): T_(R=3.83)min.

Example 76

Allyl(6aS)-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(72)

A solution of4-(4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (71) (50.0 mg, 0.06 mmol) in N,N—dimethylformamide (4 mL) wascharged with N,N—dimethylpyridin-4-amine (34.5 mg, 0.18 mmol) andN—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (28.7 mg,0.15 mmol) and it was stirred for 30 min at room temperature. To thereaction mixture, aniline (6.9 g, 0.07 mmol) was then added and thesolution was stirred for further 18 h. The reaction mixture was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (10 mL)and loaded with brine (40 mL). The aqueous phase was extracted withethyl acetate (2×30 mL). The combined organic extracts were concentratedin vacuo. The resulting residue was purified by column chromatography(silica), eluting with acetone/dichloromethane (from 0% to 40%), to givethe title compound (43.0 mg, 77%) as a cream solid, (mixture ofdiastereomers).

¹H NMR (400 MHz, CD₃OD) δ 7.66 (dd, J=8.7, 1.1 Hz, 2H), 7.61-7.57 (m,2H), 7.55-7.52 (m, 2H), 7.37-7.32 (m, 3H), 7.27 (d, J=1.5 Hz, 1H), 7.19(d, J=1.8 Hz, 1H), 7.16 (s, 1H), 7.14-7.09 (m, 1H), 6.93-6.84 (m, 2H),6.21 (d, J=10.1 Hz, 1H), 6.01-5.71 (m, 1H), 5.17-4.97 (m, 2H), 4.64-4.45(m, 2H), 4.24-4.04 (m, 3H), 3.97 (s, 3H), 3.90 (s, 3H), 3.87 (br. s.,5H), 3.64-3.41 (m, 3H), 3.13-3.01 (m, 1H), 2.54 (t, J=6.9 Hz, 2H),2.19-2.15 (m, 2H), 1.83-1.48 (m, 12H); ¹³C NMR (100 MHz, CD₃OD) δ 162.5,162.2, 151.0, 140.0, 137.7, 133.5, 132.2, 129.8, 127.7, 126.8, 126.1,125.0, 124.7, 124.6, 123.3, 122.4, 122.1, 115.7, 111.9, 106.4, 101.3,85.6, 69.6, 69.5, 64.2, 63.2, 56.7, 40.2, 37.2, 37.0, 36.9, 31.8, 31.7,30.7, 29.6, 26.6, 26.5, 24.2, 24.0, 20.6, 20.5, 19.1; MS (ES+): m/z=928(M+H)⁺; LCMS (Method B): T_(R=4.33) min.

Example 77

(S)—4-(4-((2-Methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-N—(4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)-1H-pyrrole-2-carboxamide(73)

To a solution of allyl(6aS)-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(72) (43 mg, 0.05 mmol) in dichloromethane (2 mL) was sequentially addedtetrakis(triphenylphosphine)palladium(0) (3 mg, 5 mol %), andpyrrolidine (5 μL, 0.06 mmol). The reaction mixture was stirred at roomtemperature for 30 min. The reaction mixture concentrated in vacuo andsubjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%) to give the title compound (11.0 mg, 30%) as a cream solid.

¹H NMR (400 MHz, (CD₃)₂CO) δ 9.20 (s, 1H), 9.12 (s, 1H), 9.10 (s, 1H),7.98 (d, J=5.5 Hz, 1H), 7.80 (d, J=7.8 Hz, 2H), 7.75 (d, J=8.6 Hz, 2H),7.49 (d, J=8.6 Hz, 2H), 7.36-7.30 (m, 5H), 7.22 (S, 1H), 7.10-7.04 (m,1H), 6.92 (d, J=1.6 Hz, 1H), 6.78 (s, 1H), 4.20-4.04 (m, 2H), 4.00 (s,3H), 3.91 (s, 3H), 3.86 (s, 3H), 3.81-3.70 (m, 2H), 3.23-3.10 (1H), 2.52(t, J=7.2 Hz, 2H), 2.20-2.12 (m, 3H), 2.02-1.90 (m, 1H), 1.88-1.56 (m,4H); ¹³C NMR (100 MHz, (CD₃)₂CO) δ 159.8, 159.7, 139.6, 131.8, 137.5,130.0, 128.5, 126.6, 125.1, 124.7, 123.2, 123.1, 122.7, 120.2, 119.7,118.8, 111.8, 109.9, 104.2, 68.0, 56.8, 55.4, 49.6, 39.1, 36.1, 35.8,32.2, 28.4, 25.0, 24.1, 22.9, 18.2, 18.0; MS (ES+): m/z=742 (M+H)⁺; LCMS(Method B): T_(R=3.78) min. HRMS (EI, m/z): calculated for C₄₂H₄₃N₇O₆(M+1)⁺ 742.3348, observed 742.3328.

Example 78

Allyl(6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(74)

A solution of4-(4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (71) (340.0 mg, 0.40 mmol) in N,N—dimethylformamide (6 mL) wascharged with N,N—dimethylpyridin-4-amine (146.0 mg, 1.19 mmol) andN—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (190.7 mg,0.99 mmol) and it was stirred for 30 min at room temperature. To thereaction mixture, p-phenylenediamine (52 mg, 0.48 mmol) was then addedand the solution was stirred for further 18 h. The reaction mixture wasquenched with a saturated aqueous solution of sodium hydrogen carbonate(20 mL) and loaded with brine (70 mL). The aqueous phase was extractedwith ethyl acetate (2×50 mL). The combined organic extracts wereconcentrated in vacuo. The resulting residue was purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%), to give the title compound (320 mg, 85%) as a cream solid,(mixture of diastereomers).

¹H NMR (400 MHz, (CD₃)₂SO) δ 9.93 (s, 1H), 9.80 (s, 1H), 9.50 (s, 1H),7.70 (d, J=8.6 Hz, 2H), 7.48 (d, J=8.6 Hz, 2H), 7.40 (d, J=1.2 Hz, 1H),7.36-7.28 (m, 3H), 7.23 (s, 1H), 7.06 (d, J=2.2 Hz, 1H), 6.95 (br. s.,1H), 6.92 (br. s., 0.4H), 6.80 (s, 0.6H), 6.53 (d, J=8.6 Hz, 2H), 6.03(d, J=9.8 Hz, 0.6H), 5.94 (d, J=10.0 Hz, 0.4H), 5.84-5.67 (m, 1H), 5.05(br. s., 2H), 4.67-4.37 (m, 2H), 4.16-3.93 (m, 3H), 3.89 (s, 3H),3.85-3.80 (m, 6H), 3.79-3.73 (m, 1H), 3.57-3.45 (m, 1H), 2.98-2.83 (m,1H), 2.44 (t, J=6.7 Hz, 2H), 2.10-1.98 (m, 2H), 1.78-1.32 (m, 12H); ¹³CNMR (100 MHz, (CD₃)₂SO) δ 169.3, 169.2, 168.6, 168.5, 160.0 159.7,149.1, 145.3, 137.5, 133.2, 130.1, 128.7, 127.6, 127.1, 125.1, 124.8,123.2, 122.5, 122.3, 122.2, 120.9, 119.2, 116.9, 114.5, 114.2, 110.1,105.1, 68.5, 66.0, 63.2, 56.2, 55.3, 38.7, 38.6, 36.8, 32.2, 32.1, 30.6,25.4, 23.1, 18.3; MS (ES+): m/z=943 (M+H)⁺, 941 (M−H)⁻; LCMS (Method B):T_(R=3.43) min.

Example 79

(S)—N—(4-aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(41)

To a solution of allyl(6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(74) (250 mg, 0.265 mmol) in dichloromethane (3 mL) was sequentiallyadded tetrakis(triphenylphosphine)palladium(0) (15 mg, 5 mol %), andpyrrolidine (26 mL, 0.32 mmol). The reaction mixture was stirred at roomtemperature for 30 min. The reaction mixture concentrated in vacuo andsubjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%) to give the title compound (118 mg, 59%) as a cream solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 9.88-9.96 (m, 1H), 9.81 (s, 2H), 9.50 (s,1H), 8.32 (br s, 2H), 8.00 (d, J=5.7 Hz, 1H), 7.67-7.73 (m, 2H), 7.48(d, J=8.6 Hz, 2H), 7.39 (d, J=1.8 Hz, 1H), 7.31-7.35 (m, 2H), 7.30 (d,J=1.6 Hz, 1H), 7.27 (s, 1H), 7.22 (d, J=1.5 Hz, 1H), 6.96 (d, J=1.6 Hz,1H), 6.80 (s, 1H), 6.51-6.55 (m, 2H), 4.09-4.17 (m, 1H), 3.99-4.05 (m,1H), 3.90-3.97 (m, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H),3.68-3.72 (m, 1H), 3.05-3.16 (m, 2H), 2.44 (t, J=7.3 Hz, 2H), 2.02-2.07(m, 2H), 1.81-1.91 (m, 1H), 1.68-1.78 (m, 2H), 1.56 (d, J=4.9 Hz, 2H);¹³C NMR (100 MHz, (CD₃)₂SO) δ 168.8, 166.3, 164.7, 159.5, 159.2, 150.2,147.1, 144.7, 139.8, 137.0, 129.6, 128.2, 126.1, 124.6, 124.3, 122.0,121.8, 120.4, 120.2, 118.8, 113.7, 111.3, 109.6, 104.7, 67.7, 67.2,55.6, 51.1, 49.2, 38.5, 36.2, 36.1, 35.4, 31.8, 30.2, 24.7, 23.7, 22.5,17.7; MS (ES+): m/z=757 (M+H)⁺; LCMS (Method A): T_(R=5.80) min.

Example 80

Allyl(6aS)-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(p-tolylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(75)

A solution of4-(4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (71) (50.0 mg, 0.06 mmol) in N,N—dimethylformamide (4 mL) wascharged with N,N—dimethylpyridin-4-amine (34.5 mg, 0.18 mmol) andN—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (28.7 mg,0.15 mmol) and it was stirred for 30 min at room temperature. To thereaction mixture, p-toluidine (7.7 mg, 0.07 mmol) was then added and thesolution was stirred for further 18 h. The reaction mixture was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (10 mL)and washed with a saturated aqueous solution of sodium chloride (40 mL).The aqueous phase was extracted with ethyl acetate (2×30 mL). Thecombined organic extracts were concentrated in vacuo. The resultingresidue was purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 40%), to give the title compound (40mg, 71%) as a cream solid, (mixture of diastereomers).

¹H NMR (400 MHz, CD₃OD) δ 7.62-7.57 (m, 2H), 7.56-7.52 (m, 3H),7.52-7.50 (m, 1H), 7.30 (d, J=2.0 Hz, 1H), 7.27 (d, J=1.8 Hz, 1H),7.21-7.19 (m, 1H), 7.18-7.15 (m, 3H), 6.93-6.85 (m, 2H), 6.21 (d, J=10.1Hz, 0.7H), 5.99 (d, J=10.6 Hz, 0.3H), 5.75 (br. s., 1H), 5.09 (br. s.,2H), 4.64-4.48 (m, 2H), 4.23-4.03 (m, 3H), 3.97 (s, 3H), 3.91 (s, 3H),3.89-3.79 (m, 5H), 3.63-3.44 (m, 2H), 3.14-3.03 (m, 1H), 2.54 (t, J=6.9Hz, 2H), 2.33 (s, 3H), 2.14-2.02 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ1.84-1.48 (m, 12H); 162.4, 151.0, 137.7, 137.3, 134.8, 133.5, 132.3,130.2, 127.8, 126.7, 126.1, 124.7, 124.6, 122.4, 122.2, 115.7, 111.7,106.4, 69.6, 56.7, 54.8, 40.2, 37.1, 36.9, 31.8, 31,66, 30.7, 29.6,26.5, 24.2, 24.0, 21.0, 20.6, 20.5, 19.1; MS (ES+): m/z=943 (M+H)⁺; LCMS(Method A): t_(R=4.32) min.

Example 81

(S)—4-(4-((2-Methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-N—(4-(1-methyl-5-(p-tolylcarbamoyl)-1H-pyrrol-3-yl)phenyl)-1H-pyrrole-2-carboxamide(76)

To a solution of allyl(6aS)-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(p-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(75) (60 mg, 0.07 mmol) in dichloromethane (2 mL) was sequentially addedtetrakis(triphenylphosphine)palladium(0) (4 mg, 5 mol %), andpyrrolidine (6.8 mL, 0.08 mmol). The reaction mixture was stirred atroom temperature for 30 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with acetone/dichloromethane (from 0%to 40%) to give the title compound (30 mg, 57%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 2H), 8.12 (s, 1H), 7.86 (d, J=5.5 Hz,1H), 7.50 (d, J=8.2 Hz, 2H), 7.44 (d, J=8.6 Hz, 2H), 7.39 (s, 1H), 7.28(d, J=8.6 Hz, 2H), 7.13-7.07 (m, 3H), 6.96 (d, J=2.3 Hz, 2H), 6.74 (s,1H), 6.48 (s, 1H), 3.98 (t, J=5.9 Hz, 2H), 3.92 (s, 3H), 3.80 (d, J=4.7Hz, 6H), 3.76-3.66 (m, 2H), 3.23-3.10 (m, 1H), 2.47-2.38 (m, 2H), 2.29(s, 3H), 2.18-2.10 (m, 2H), 2.09-197 (m, 2H), 1.95-1.58 (m, 4H); ¹³C NMR(100 MHz, CDCl₃) δ 170.1, 167.6, 163.5, 160.0, 159.9, 150.7, 147.9,140.3, 139.9, 136.1, 135.6, 133.5, 130.3, 129.4, 126.6, 125.2, 125.0,123.1, 121.6, 121.2, 120.8, 120.3, 119.9, 110.1, 109.5, 68.1, 61.1,56.0, 50.7, 49.7, 39.8, 36.9, 36.6, 24.9, 24.4, 22.9, 20.9, 18.3; MS(ES+): m/z=756 (M+H)⁺; LCMS (Method A): T_(R=3.83) min. HRMS (EI, m/z):calculated for C₄₃H₄₅N₇O₆ (M+1)⁺ 756.3504, observed 756.3489.

Example 82

4-(4-((tert-Butoxycarbonyl)amino)phenyl)-1-methyl-1H-pyrrole-2-carboxylicAcid (77)

To a solution of methyl4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(70) (800 mg, 2.42 mmol) in 1,4-dioxane (15 mL) was added an aqueoussolution of sodium hydroxide (1 M, 15 mL, 10 mmol). The reaction mixturewas stirred at room temperature for 18 h and was then concentrated invacuo, after which water (100 mL) was added and the aqueous layer wasacidified to pH=4 with an aqueous solution of acetic acid (5 M, 25 mL).The aqueous layer was then extracted with ethyl acetate (2×100 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated to give the title compound (700 mg, 91%) as a white solid.The product was carried through to the next step without any furtherpurification.

¹H NMR (400 MHz, CD₃OD) δ 7.45-7.42 (m, 2H), 7.38-7.34 (m, 2H), 7.25 (d,J=2.0 Hz, 1H), 7.19 (d, J=2.0 Hz, 1H), 3.92 (s, 3H), 1.52 (s, 9H); ¹³CNMR (100 MHz, CD₃OD) δ 172.1, 164.5, 155.5, 138.3, 136.6, 130.8, 129.8,127.8, 126.3, 124.9, 124.4, 120.4, 116.3, 116.1, 81.1, 61.8, 37.4, 28.8;MS (ES+): m/z=315 (M−H)⁻; LCMS (Method A): T_(R=3.68) min.

Example 83

Methyl4-(4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)benzoate(78)

A solution of4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (77) (250.0 mg, 0.79 mmol) in N,N—dimethylformamide (6 mL) wascharged with N,N—dimethylpyridin-4-amine (291.0 mg, 2.38 mmol) andN—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (380.5 mg,1.98 mmol) and it was stirred for 30 min at room temperature. To thereaction mixture, methyl 4-aminobenzoate (132.0 mg, 0.87 mmol) was thenadded and the solution was stirred for further 18 h. The reactionmixture was quenched with a saturated aqueous solution of sodiumhydrogen carbonate (20 mL) and loaded with brine (80 mL). The aqueousphase was extracted with ethyl acetate (2×60 mL). The combined organicextracts were concentrated in vacuo. The resulting residue was purifiedby column chromatography (silica), eluting with ethylacetate/dichloromethane (from 0% to 40%), to give the title compound (61mg, 17%) as a brown solid.

¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J=8.8 Hz, 2H), 7.81 (s, 1H), 7.61 (d,J=8.8 Hz, 2H), 7.35-7.31 (m, 2H), 7.30-7.26 (m, 2H), 6.97 (d, J=1.8 Hz,1H), 6.88 (d, J=1.8 Hz, 1H), 6.46 (s, 1H), 3.92 (s, 3H), 3.84 (s, 3H),1.46 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 166.7, 159.6, 142.4, 136.6,136.6, 130.9, 129.2, 126.0, 125.8, 125.6, 123.6, 119.0, 118.8, 109.7,52.0, 37.1, 29.7, 28.4; MS (ES+): m/z=450 (M+H)⁺; LCMS (Method A):T_(R=4.32) min.

Example 84

Methyl4-(4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)benzoate(79)

To a solution of methyl4-(4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)benzoate(78) (77.0 mg, 0.17 mmol) in dioxane and methanol (1:1) (6 mL)hydrochloric acid (4 M in 1,4-dioxane) (6 mL) was added drop wise. Thereaction mixture was stirred for 3 h and then concentrated in vacuo. Theresidue was added to a mixture of4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid(45.0 mg, 0.19 mmol), N,N—dimethylpyridin-4-amine (63.0 mg, 0.52 mmol)and N—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (82.4mg, 0.43 mmol) in N,N—dimethylformamide (4 mL) which was previouslystirred for 30 min. The resulting solution was allowed to react at roomtemperature for 18 h. The reaction mixture was quenched with a saturatedaqueous solution of sodium hydrogen carbonate (5 mL) and washed with asaturated aqueous solution of sodium chloride (80 mL). The aqueous phasewas extracted with ethyl acetate (2×30 mL). The combined organicextracts were concentrated in vacuo. The resulting residue was purifiedby column chromatography (silica), eluting with acetone/dichloromethane(from 0% to 30%), to give the title compound (89.0 mg, %) as a brownsolid.

¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.94-7.91 (m, 3H), 7.71 (s, 1H),7.66 (d, J=8.6 Hz, 2H), 7.40 (d, J=8.6 Hz, 2H), 7.27 (d, J=8.3 Hz, 2H),6.96 (s, 1H), 6.94 (s, 1H), 6.77 (br. s., 1H), 3.89 (s, 3H), 3.82 (s,3H), 3.79 (s, 3H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 166.8,162.6, 160.1, 159.9, 159.7, 142.8, 136.1, 130.8, 130.2, 126.1, 125.7,125.4, 125.0, 123.4, 123.3, 121.9, 120.6, 119.0, 110.3, 104.2, 61.7,52.0, 37.1, 36.7, 36.5, 31.5, 28.4; MS (ES+): m/z=572 (M+H)⁺; LCMS(Method A): T_(R=4.17) min.

Example 85

Allyl(6aS)-2-methoxy-3-(4-((5-((4-(5-((4-(methoxycarbonyl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(80)

To a solution of methyl4-(4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)benzoate(79) (93.0 mg, 0.16 mmol) in dioxane and methanol (1:1) (3 mL)hydrochloric acid (4 M in 1,4-dioxane) (3 mL) was added drop wise. Thereaction mixture was stirred for 4 h and then concentrated in vacuo. Theresidue was added to a mixture of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (71.0 mg, 0.13 mmol), N,N—dimethylpyridin-4-amine (47.6 mg,0.39 mmol) and N—(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (61.3 mg, 0.32 mmol) in N,N—dimethylformamide (4 mL) whichwas previously stirred for 30 min. The resulting solution was allowed toreact at room temperature for 18 h. The reaction mixture was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (5 mL)and washed with a saturated aqueous solution of sodium chloride (70 mL).The aqueous phase was extracted with ethyl acetate (2×60 mL). Thecombined organic extracts were concentrated in vacuo. The resultingresidue was purified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 30%), to give the title compound(80.0 mg, 62%) as an orange solid (mixture of diastereomers).

¹H NMR (400 MHz, CD₃OD) δ 7.91-7.86 (m, 4H), 7.75-7.69 (m, 2H),7.53-7.47 (m, 2H), 7.46-7.41 (m, 2H), 7.27 (d, J=2.0 Hz, 1H), 7.21 (d,J=1.5 Hz, 1H), 7.05 (s, 1H), 6.84-6.73 (m, 2H), 6.09 (d, J=10.3 Hz, 1H),5.79-5.59 (m, 1H), 4.96 (br. s., 2H), 4.43-4.36 (m, 2H), 4.08-4.03 (m,1H), 4.03-3.03 (m, 3H), 3.88 (s, 3H), 3.81-3.70 (m, 11H), 3.66-3.58 (m,1H), 3.02-2.94 (m, 1H), 2.48-2.41 (m, 2H) 2.10-2.04 (m, 2H), 1.69-1.42(m, 12H); ¹³C NMR (100 MHz, CD₃OD) δ 177.5, 168.3, 145.1, 141.6, 137.8,132.1, 131.5, 127.4, 127.3, 126.1, 125.9, 124.7, 124.6, 123.2, 122.4,120.6, 112.4, 112.2, 111.3, 70.6, 69.5, 67.0, 63.1, 56.6, 56.1, 54.8,52.5, 43.7, 37.3, 37.0, 36.9, 34.6, 31.7, 29.5, 26.6, 24.1, 20.5, 19.2;MS (ES+): m/z=986 (M+H)⁺; LCMS (Method A): T_(R=4.28) min.

Example 86

Methyl(S)—4-(4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)benzoate(81)

To a solution of allyl(6aS)-2-methoxy-3-(4-((5-((4-(5-((4-(methoxycarbonyl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(80) (80 mg, 0.08 mmol) in dichloromethane (3 mL) was sequentially addedtetrakis(triphenylphosphine)palladium(0) (4.7 mg, 5 mol %), andpyrrolidine (5.6 mL, 0.07 mmol). The reaction mixture was stirred atroom temperature for 30 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with acetone/dichloromethane (from 0%to 40%) to give the title compound (14 mg, 22%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 8.71 (s, 1H), 8.21 (S, 1H), 8.11 (s, 1H), 7.98(d, J=8.6 Hz, 2H), 7.86 (d, J=5.9 Hz, 1H), 7.76 (d, J=8.6 Hz, 2H), 7.43(d, J=8.6 Hz, 2H), 7.39 (s, 1H), 7.24 (d, J=8.6 Hz, 2H), 7.09 (s, 1H),7.02 (s, 1H), 6.98 (s, 1H), 6.73 (s, 1H), 6.48 (s, 1H), 3.99 (t, J=5.5Hz, 2H), 3.94 (s, 3H), 3.88 (s, 3H), 3.79 (d, J=50.1 Hz, 6H), 3.77-3.66(m, 2H), 3.23-3.11 (m, 1H), 2.45 (t, J=6.8 Hz, 2H), 2.19-2.10 (m, 2H),2.06-1.70 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 174.7, 170.2, 1668, 161.6,160.0, 157.5, 154.2, 147.9, 146.1, 142.9, 139.9, 136.1, 132.2, 130.7,126.1, 125.6, 125.6, 125.2, 124.9, 123.2, 120.9, 120.1, 119.1, 87.6,81.4, 62.6, 56.0, 52.0, 39.8, 37.1, 36.6, 31.2, 24.4, 23.0, 18.3; MS(ES+): m/z=800 (M+H)⁺; LCMS (Method A): T_(R=3.78) min. HRMS (EI, m/z):calculated for C₄₄H₄₅N₇O₈ (M+1)⁺ 800.3402, observed 800.3387.

Example 87

4-Bromo-1-methyl-1H-imidazole-2-carboxylic Acid (83)

To a solution of methyl 4-bromo-1-methyl-1H-imidazole-2-carboxylate (200mg, 0.91 mmol) in 1,4-dioxane (8 mL) was added an aqueous solution ofsodium hydroxide (1 M, 8 mL, 10 mmol). The reaction mixture was stirredat room temperature for 2 h and was then concentrated in vacuo, afterwhich water (80 mL) was added and the aqueous layer was acidified topH=4 with an aqueous solution of acetic acid (5 M, 15 mL). The aqueouslayer was then extracted with ethyl acetate (2×60 mL). The combinedorganic extracts were dried over sodium sulfate, filtered andconcentrated to give the title compound (190 mg, 93%) as a yellow solid.The product was carried through to the next step without any furtherpurification.

¹H NMR (400 MHz, CDCl₃) δ 10.90 (br. S., 1H), 7.44 (s, 1H), 3.66 (s,3H); ¹³C NMR (100 MHz, CDCl₃) δ 175.7, 154.4, 137.6, 125.3, 34.1; MS(ES+): m/z=207 (M+H)⁺, 205 (M−H); LCMS (Method B): T_(R=1.80) min.

Example 88

4-Bromo-1-methyl-N—phenyl-1H-imidazole-2-carboxamide (84)

To a solution of 4-bromo-1-methyl-1H-imidazole-2-carboxylic acid (83)(190.0 mg, 0.92 mmol) in N,N—dimethylformamide (3 mL) was charged withN,N—dimethylpyridin-4-amine (338.0 mg, 2.77 mmol) andN—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (442.0 mg,2.30 mmol) and it was stirred for 30 min at room temperature. To thereaction mixture, aniline (90.2 mg, 1.01 mmol) was then added and thesolution was stirred for further 20 h. The reaction mixture was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (10 mL)and washed with a saturated aqueous solution of sodium chloride (40 mL).The aqueous phase was extracted with ethyl acetate (2×30 mL). Thecombined organic extracts were concentrated in vacuo. The resultingresidue was purified by column chromatography (silica), eluting withethyl acetate/petroleum ether (from 0% to 40%), to give the titlecompound (60 mg, 23%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 9.05 (br. s., 1H), 7.63 (d, J=7.8 Hz, 2H),7.35 (t, J=7.6 Hz, 2H), 7.13 (t, J=8.0 Hz, 1H), 6.98 (s, 1H), 4.08 (s,3H); ¹³C NMR (100 MHz, CDCl₃) δ 155.8, 138.6, 137.2, 129.1, 125.3,124.5, 119.8, 36.1; MS (ES+): m/z=280 (M+H)⁺, 205 (M−H)⁻; LCMS (MethodB): T_(R=3.97) min.

Example 89

tert-Butyl(4-(1-methyl-2-(phenylcarbamoyl)-1H-imidazol-4-yl)phenyl)carbamate (85)

To a solution of 4-bromo-1-methyl-N—phenyl-1H-imidazole-2-carboxamide(84) (60.0 mg, 0.21 mmol) in N,N—dimethylformamide (3 mL) and water (2mL) tert-butyl (4-(dihydroxyamino)phenyl)carbamate (96.0 mg, 0.30 mmol),caesium carbonate (209 mg, 0.64 mmol), andtetrakis(triphenylphosphine)palladium (13 mg, mol 5%) were added. Thereaction mixture was purged with nitrogen for 5 min and the reaction wascarried out in a microwave reactor at 100° C. for 2 h. The mixture wasfiltered through a celite pad. The pad was washed with ethyl acetate(100 mL) and the resulting organic solution was concentrated in vacuo.The residue was purified by column chromatography (silica), eluting withethyl acetate/hexane (from 0% to 50%), to give the title compound (40mg, 47%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 9.33 (br. s., 1H), 7.74-7.66 (m, 4H),7.44-7.33 (m, 4H), 7.22 (d, J=1.6 Hz, 1H), 7.16-7.11 (m, 1H), 6.61 (s,1H), 4.10 (s, 3H), 1.53 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 157.2,140.1, 138.6, 137.7, 137.6, 129.0, 125.7, 124.2, 121.4, 119.7, 119.6,118.6, 80.6, 35.9, 28.3; MS (ES+): m/z=393 (M+H)⁺; LCMS (Method B):T_(R=4.40) min

Example 90

tert-Butyl(1-methyl-5-((4-(1-methyl-2-(phenylcarbamoyl)-1H-imidazol-4-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)carbamate(86)

To a solution of tert-butyl(4-(1-methyl-2-(phenylcarbamoyl)-1H-imidazol-4-yl)phenyl)carbamate (85)(40.0 mg, 0.10 mmol) in dioxane and methanol (1:1) (2 mL) hydrochloricacid (4 M in 1,4-dioxane) (2 mL) was added drop wise. The reactionmixture was stirred for 3 h and then concentrated in vacuo. The residuewas added to a mixture of4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid(30.0 mg, 0.12 mmol), N,N—dimethylpyridin-4-amine (38.0 mg, 0.31 mmol)and N—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (49.0mg, 0.25 mmol) in N,N—dimethylformamide (3 mL) which was previouslystirred for 30 min. The resulting solution was allowed to react at roomtemperature over 20 h. The reaction mixture was quenched with asaturated aqueous solution of sodium hydrogen carbonate (10 mL) andwashed with a saturated aqueous solution of sodium chloride (50 mL). Theaqueous phase was extracted with ethyl acetate (2×40 mL). The combinedorganic extracts were concentrated in vacuo. The resulting residue waspurified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 30%), to give the title compound (43mg, 82%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 9.34 (s, 1H), 7.75-7.67 (m, 4H), 7.58 (d,J=8.2 HZ, 2H), 7.38-7.33 (m, 2H), 7.21 (s, 1H), 7.13 (t, J=8.0 Hz, 1H),7.06 (br. s., 1H), 6.85 (br. s., 1H), 6.64 (br. s., 1H), 6.45 (br. s.,1H), 4.09 (s, 3H), 3.89 (s, 3H), 1.50 (s, 9H); ¹³C NMR (100 MHz, CDCl₃)δ 159.5, 157.0, 140.0, 137.6, 137.4, 129.0, 125.6, 124.3, 123.4, 121.9,121.6, 120.1, 119.9, 119.8, 118.8, 110.4, 104.0, 36.7, 35.9, 28.4, 28.3;MS (ES+): m/z=515 (M+H)⁺; LCMS (Method B): T_(R=4.33) min.

Example 91

Allyl(6aS)-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-2-(phenylcarbamoyl)-1H-imidazol-4-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(87)

To a solution of tert-butyl(1-methyl-5-((4-(1-methyl-2-(phenylcarbamoyl)-1H-imidazol-4-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)carbamate(86) (41.5 mg, 0.10 mmol) in dioxane and methanol (1:1) (2 mL)hydrochloric acid (4 M in 1,4-dioxane) (2 mL) was added drop wise. Thereaction mixture was stirred for 4 h and then concentrated in vacuo. Theresidue was added to a mixture of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (64.0 mg, 0.12 mmol), N,N—dimethylpyridin-4-amine (37.0 mg, 0.3mmol) and N—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(48.0 mg, 0.25 mmol) in N,N—dimethylformamide (3 mL) which waspreviously stirred for 30 min. The resulting solution was left to reactat room temperature for 18 h. The reaction mixture was quenched with asaturated aqueous solution of sodium hydrogen carbonate (3 mL) andwashed with a saturated aqueous solution of sodium chloride (40 mL). Theaqueous phase was extracted with ethyl acetate (2×30 mL). The combinedorganic extracts were concentrated in vacuo. The resulting residue waspurified by column chromatography (silica), eluting withacetone/dichloromethane (from 0% to 40%), to give the title compound (33mg, 78%) as a brown viscous oil.

¹H NMR (400 MHz, CDCl₃) δ 9.34 (s, 1H), 7.73 (d, J=6.2 Hz, 3H), 7.68 (d,J=7.8 Hz, 2H), 7.36 (t, J=7.8 Hz, 2H), 7.25 (d, J=8.2 Hz, 2H), 7.19-7.08(m, 3H), 6.78 (br. s., 1H), 6.18 (br. s., 1H), 6.02-5.75 (m, 1H),5.11-5.01 (m, 2H), 4.67-4.29 (m, 2H), 4.11 (s, 4H), 3.89-3.77 (m, 9H),3.6.2 (br. s., 3H), 3.12-3.05 (m, 1H), 2.52-2.37 (m, 2H), 2.19-2.14 (m,2H), 1.76-1.46 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 188.2, 157.0, 148.3,147.2, 138.8, 138.7, 138.6, 137.6, 135.1, 134.4, 132.0, 131.8, 129.2,129.0, 127.6, 125.4, 124.2, 122.3, 121.6, 121.4, 120.4, 119.8, 118.3,115.2, 110.9, 107.9, 99.9, 89.8 84.2, 68.8, 56.1, 54.2, 53.8, 53.4,50.4, 42.6, 39.0, 36.8, 35.9, 31.0, 30.6, 29.2, 25.2, 22.9, 19.6, 18.1;MS (ES+): m/z=930 (M+H)⁺; LCMS (Method B): T_(R=4.42) min

Example 92

(S)—4-(4-(4-(4-((2-Methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-N—phenyl-1H-imidazole-2-carboxamide(88)

To a solution of allyl(6aS)-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-2-(phenylcarbamoyl)-1H-imidazol-4-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(87) (30.0 mg, 0.03 mmol) in dichloromethane (2 mL) was sequentiallyadded tetrakis(triphenylphosphine)palladium(0) (1.9 mg, 5 mol %), andpyrrolidine (3.5 mL, 0.04 mmol). The reaction mixture was stirred atroom temperature for 30 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%) to give the title compound (12 mg, 50%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 9.34 (s, 1H), 8.04 (s, 1H), 7.91 (s, 1H), 7.89(d, J=5.9 Hz, 1H), 7.76-7.71 (m, 2H), 7.68 (d, J=8.2 Hz, 2H), 7.66-7.61(m, 2H), 7.43 (s, 1H), 7.36 (t, J=7.8 Hz, 2H), 7.22 (s, 1H), 7.16-7.09(m, 2H), 6.79 (s. 1H), 6.52 (s, 1H), 4.13-4.07 (m, 5H), 3.88 (s, 3H),3.85 (s, 3H), 3.80-3.69 (m, 2H), 3.26-3.15 (m, 1H), 2.50 (t, J=6.83 Hz,2H), 2.21 (t, J=6.2 Hz, 2H), 2.10-2.02 (m, 6H); ¹³C NMR (100 MHz, CDCl₃)δ 169.9, 167.6, 163.5, 159.7, 157.0, 150.7, 148.0, 140.0, 139.9, 138.6,137.6, 137.6, 129.0, 128.8, 125.5, 124.3, 123.2, 121.6, 121.5, 121.4,120.3, 119.9, 119.8, 111.8, 110.4, 104.1, 68.1, 56.1, 49.7, 39.8, 36.7,35.9, 33.0, 29.3, 24.5, 22.9, 18.3; MS (ES+): m/z=743 (M+H)⁺; LCMS(Method B): T_(R=3.75) min.

Example 93

Methyl4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(89)

To a solution of methyl4-(4-((tert-butoxycarbonyl)amino)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(70) (700 mg, 2.12 mmol) in 1,4-dioxane and methanol (1:1) (8 mL)hydrochloric acid (4 M in 1,4-dioxane) (8 mL) was added drop wise. Thereaction mixture was stirred for 3 h and then concentrated in vacuo. Theresidue was added to a mixture of4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazole-2-carboxylic acid(613.1 mg, 2.54 mmol), N,N—dimethylpyridin-4-amine (777.0 mg, 6.36 mmol)and N—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.02g, 5.30 mmol) in N,N—dimethylformamide (8 mL) which was previouslystirred for 30 min. The resulting solution was allowed to react at roomtemperature for 18 h. The reaction mixture was quenched with a saturatedaqueous solution of sodium hydrogen carbonate (20 mL) and washed with asaturated aqueous solution of sodium chloride (130 mL). The aqueousphase was extracted with ethyl acetate (2×60 mL). The combined organicextracts were concentrated in vacuo. The resulting residue was purifiedby column chromatography (silica), eluting with acetone/dichloromethane(from 0% to 30%), to give the title compound (580 mg, 60%) as a brownsolid.

¹H NMR (400 MHz, CDCl₃) δ 8.99 (br. s., 1H), 7.90 (br. s., 1H), 7.55 (d,J=8.6 Hz, 2H), 7.38 (d, J=8.6 Hz, 2H), 7.17 (br. s., 1H), 7.14 (d, J=2.3Hz, 1H), 6.99 (d, J=1.9 Hz, 1H), 4.01 (s, 3H), 3.90 (s, 3H), 3.80 (s,3H), 1.44 (br. s., 9H); ¹³C NMR (100 MHz, CDCl₃) δ 171.1, 161.6, 156.4,136.8, 135.6, 130.5, 126.0, 125.4, 123.5, 123.0, 120.0, 114.6, 112.7,80.8, 51.1, 36.9, 35.8, 28.2; MS (ES+): m/z=454 (M+H)⁺; LCMS (Method B):T_(R=4.28) min.

Example 94

Allyl(6aS)-2-methoxy-3-(4-((2-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(90)

To a solution of methyl4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(89) (77.0 mg, 0.17 mmol) in dioxane and methanol (1:1) (2 mL)hydrochloric acid (4 M in 1,4-dioxane) (2 mL) was added drop wise. Thereaction mixture was stirred for 4 h and then concentrated in vacuo. Theresidue was added to a mixture of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (75.5 mg, 0.14 mmol), N,N—dimethylpyridin-4-amine (52.0 mg,0.42 mmol) and N—(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (67.6 mg, 0.35 mmol) in N,N—dimethylformamide (4 mL) whichwas previously stirred for 30 min. The resulting solution was allowed toreact at room temperature for 18 h. The reaction mixture was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (5 mL)and loaded with brine (50 mL). The aqueous phase was extracted withethyl acetate (2×30 mL). The combined organic extracts were concentratedin vacuo. The resulting residue was purified by column chromatography(silica), eluting with acetone/dichloromethane (from 0% to 30%), to givethe title compound (97.4 mg, 66%) as a yellow oil (mixture ofdiastereomers).

¹H NMR (400 MHz, CDCl₃) δ 9.01-8.93 (m, 1H), 8.11-7.99 (m, 1H),7.62-7.58 (m, 2H), 7.45 (d, J=8.6 Hz, 2H), 7.42 (s, 1H), 7.20-7.16 (m,2H), 7.06 (d, J=1.9 Hz, 1H), 6.60 (s, 1H), 6.18 (d, J=1.5 Hz, 0.7H),6.00 (d, J=9.8 Hz, 0.3H), 5.82-5.63 (m, 1H), 5.12-4.98 (m, 2H),4.68-4.43 (m, 2H), 4.33-4.09 (m, 4H), 4.06 (s, 3H), 3.95 (s, 3H), 3.90(s, 3H), 3.85-3.82 (m, 4H), 3.66-3.54 (m, 1H), 3.51-3.42 (m, 1H),3.14-3.00 (m, 1H), 2.59 (t, J=7.0 Hz, 2H), 2.28-2.19 (m, 2H),1.78-1.5-(m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 180.4, 162.9, 161.6,156.5, 149.4, 135.6, 132.0, 130.7, 126.0, 125.6, 123.5, 123.1, 120.0,114.6, 112.5, 101.6, 97.4, 84.2, 76.7, 68.0, 64.2, 56.1, 55.5, 51.1,38.8, 36.9, 35.8, 30.7, 25.2, 23.2, 22.9, 19.6, 18.1, 14.2; MS (ES+):m/z=868 (M+H)⁺; LCMS (Method B): t_(R=4.05) min.

Example 95

4-(4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicAcid (91)

To a solution of allyl(6aS)-2-methoxy-3-(4-((2-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(90) (158.0 mg, 0.18 mmol) in 1,4-dioxane (6 mL) was added an aqueoussolution of sodium hydroxide (1 M, 6 mL, 6 mmol). The reaction mixturewas stirred at room temperature for 18 h and was then concentrated invacuo, after which water (60 mL) was added and the aqueous layer wasacidified to pH=4 with an aqueous solution of acetic acid (5 M, 5 mL).The aqueous layer was then extracted with ethyl acetate (2×40 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated to give the title compound (100.0 mg, 64%) as a creamsolid. The product was carried through to the next step without anyfurther purification (mixture of diastereomers).

¹H NMR (400 MHz, (CD₃)₂CO) δ 7.66 (d, J=7.8 Hz, 2H), 7.58 (br. s., 1H),7.53-7.48 (m, 2H), 7.37 (s, 1H), 7.21 (s, 1H), 7.11 (s, 1H), 6.95 (s,1H), 6.19 (d, J=10.1 Hz, 0.7H), 6.03 (d, J=9.8 Hz, 0.3H), 5.80 (br. s.,1H), 5.15-5.03 (m, 2H), 4.65-4.43 (m, 2H), 4.26-4.08 (m, 4H), 4.02-3.90(m, 6H), 3.86 (s, 3H), 3.62-3.51 (m, 2H), 3.42 (br. s., 1H), 3.02-2.95(m, 1H), 2.66-2.53 (m, 2H), 2.22 (m, 2H), 1.74-1.45 (m, 12H); ¹³C NMR(100 MHz, (CD₃)₂CO) δ 161.5, 154.9, 149.4, 132.7, 126.5, 126.3, 125.8,125.1, 124.9, 119.8, 119.5, 116.3, 114.5, 114.2, 110.6, 104.2, 68.2,65.8, 64.9, 55.4, 55.2, 38.4, 36.1, 34.9, 30.6, 26.9, 25.3, 24.5, 23.0,19.4, 18.2; MS (ES+): m/z=854 (M+H)⁺; LCMS (Method B): T_(R=3.97) min.

Example 96

Allyl(6aS)-2-methoxy-3-(4-((1-methyl-2-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(92)

A solution of4-(4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (91) (70.0 mg, 0.08 mmol) in N,N—dimethylformamide (4 mL) wascharged with N,N—dimethylpyridin-4-amine (30.0 mg, 0.25 mmol) andN—(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (39.0 mg,0.20 mmol) and it was stirred for 30 min at room temperature. To thereaction mixture, aniline (8.8 mg, 0.10 mmol) was then added and thesolution was stirred for further 18 h. The reaction mixture was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (10 mL)and loaded with brine (40 mL). The aqueous phase was extracted withethyl acetate (2×30 mL). The combined organic extracts were concentratedin vacuo. The resulting residue was purified by column chromatography(silica), eluting with acetone/dichloromethane (from 0% to 40%), to givethe title compound (52 mg, 69%) as a yellow oil (mixture ofdiastereomers).

¹H NMR (400 MHz, CDCl₃) δ 7.63-7.60 (m, 2H), 7.43-7.37 (m, 2H), 7.33 (t,J=7.8 Hz, 2H), 7.25-7.22 (m, 2H), 7.18-7.14 (m, 3H), 7.13-7.07 (m, 1H),7.01 (d, J=6.2 Hz, 1H), 6.61 (br. S., 1H), 6.19 (d, J=8.6 Hz, 0.7H),6.07-5.96 (m, 0.3H), 5.74 (br. S., 1H), 5.12-5.01 (m, 2H), 4.66-4.47 (m,2H), 4.31-4.22 (m, 1H), 4.15-4.05 (m, 2H), 4.02 (s, 3H), 3.99 (s, 3H),3.88 (s, 3H), 3.82 (br. S., 1H), 3.68-3.58 (m, 1H), 3.50-3.44 (m, 2H),3.15-3.02 (m, 1H), 2.65-2.50 (m, 2H), 2.28-2.13 (m, 2H), 1.75-1.49 (m,12H); ¹³C NMR (100 MHz, CDCl₃) δ 183.0, 181.8, 169.6, 162.5, 159.9,148.3, 138.2, 137.8, 135.8, 132.8, 132.0, 129.7, 129.0, 128.9, 128.0,126.6, 125.4, 125.3, 125.2, 124.0, 123.5, 120.2, 120.0, 114.5, 111.8,111.7, 110.7, 109.5, 103.9, 97.1, 81.7, 69.8, 56.0, 52.0, 38.9, 37.0,36.5, 31.4, 30.7, 27.5, 25.2, 22.9, 21.4, 19.7, 18.1; MS (ES+): m/z=929(M+H)⁺; LCMS (Method B): t_(R=4.33) min.

Example 97

(S)—4-(4-((2-Methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-N—(4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)-1H-imidazole-2-carboxamide(93)

To a solution of allyl(6aS)-2-methoxy-3-(4-((1-methyl-2-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(92) (50.0 mg, 0.05 mmol) in dichloromethane (3 mL) was sequentiallyadded tetrakis(triphenylphosphine)palladium(0) (3.1 mg, 5 mol %), andpyrrolidine (4.5 □L, 0.06 mmol). The reaction mixture was stirred atroom temperature for 30 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%) to give the title compound (25 mg, 62%) as a cream solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 10.40 (s, 1H), 9.82 (s, 2H), 7.89 (d, J=5.5Hz, 1H), 7.73 (d, J=3.9 Hz, 2H), 7.71 (d, J=2.7 Hz, 2H), 7.54-7.48 (m,3H), 7.46 (s, 1H), 7.39 (s, 1H), 7.31 (t, J=7.8 Hz, 2H), 7.25 (s, 1H),7.07-7.00 (m, 1H), 6.78 (s, 1H), 4.13-3.99 (m, 2H), 3.95 (s, 3H), 3.88(s, 3H), 3.80 (s, 3H), 3.68-3.65 (m, 2H), 3.10-3.06 (m, 1H), 2.51 (br.s., 2H), 2.08-1.97 (m, 3H), 1.88-1.50 (m, 5H); ¹³C NMR (100 MHz,(CD₃)₂SO) δ 167.3, 165.1, 157.2, 153.9, 151.6, 147.6, 140.1, 139.8,136.6, 136.5, 134.7, 134.2, 130.7, 129.0, 126.7, 126.0, 125.1, 123.5,122.2, 120.7, 120.4, 115.0, 111.0, 90.7, 86.9, 79.2, 72.6, 56.3, 49.0,40.6, 40.4, 40.2, 40.0, 39.8, 39.5, 39.3, 36.9, 36.7, 35.7, 35.5, 31.9,25.0; MS (ES+): m/z=743 (M+H)⁺; LCMS (Method B): T_(R=3.78) min. HRMS(EI, m/z): calculated for C₄₁H₄₂N₈O₆ (M+1)⁺ 743.3300, observed 743.3291.

Example 98

4-(Benzyloxy)-3-methoxybenzaldehyde (95)

A mixture of 4-hydroxy-3-methoxybenzaldehyde (94) (200.00 g, 1.31 mol),benzyl bromide (236.07 g, 1.38 mol) and K₂CO₃ (545.02 g, 3.94 mol) inmethanol (1.2 L) was refluxed for 5 h. The reaction mixture was filteredand the filtrate was evaporated under reduced pressure to give the titlecompound (271.00 g, 85%) as a light yellow solid. The product wascarried through to the next step without any further purification.

¹H NMR (400 MHz, CDCl₃) δ 9.83 (s, 1H), 7.29-7.46 (m, 7H), 6.98 (d,J=8.1 Hz, 1H), 5.25 (s, 2H), 3.94 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ191.0, 153.6, 150.1, 136.0, 130.3, 128.7, 128.2, 127.2, 126.6, 112.3,109.3, 70.9, 56.1; MS (ES+): m/z=243 (M+H)⁺; LCMS (Method A): T_(R=7.53)min.

Example 99

4-(Benzyloxy)-5-methoxy-2-nitrobenzaldehyde (96)

To a stirred solution of 4-(benzyloxy)-3-methoxybenzaldehyde (95)(130.00 g, 536.6 mmol) in TFA (600 mL) was added KNO₃ (65.10 g, 643.9mmol, in 600 mL of TFA) dropwise at 0° C. The reaction mixture wasstirred for another hour. The reaction mixture was diluted with water(2.4 L). The precipitate was filtered and washed with cold water (2×500mL) to give the title compound (125.00 g, 81%) as a yellow solid. Theproduct was carried through to the next step without any furtherpurification.

¹H NMR (400 MHz, CDCl₃) δ 10.42 (s, 1H), 7.66 (s, 1H), 7.34-7.46 (m,6H), 5.26 (s, 2 H), 4.0 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 187.8,153.7, 151.4, 134.85, 129.0, 128.9, 128.7, 127.6, 125.7, 110.0, 108.9,71.6, 56.73; MS (ES+): m/z=286 (M−H)⁻; LCMS (Method A): T_(R=7.87) min.

Example 100

4-Hydroxy-5-methoxy-2-nitrobenzaldehyde (97)

To a stirred solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde(96) (100.00 g, 348.1 mmol) in AcOH (800 mL) was added HBr (48%, 88.02mL, 522.16 mmol). The reaction mixture was stirred at 85° C. for 1 h.The reaction mixture was diluted with H₂O (1.6 L), the precipitate wasfiltered and washed with cold water (3×100 mL) to give the titlecompound (50.00 g, 73%) as a yellow solid. The product was carriedthrough to the next step without any further purification.

¹H NMR (400 MHz, d6-DMSO) δ 11.11 (br s, 1H), 10.15 (br s, 1H), 7.50 (s,1H), 7.35 (s, 1H), 3.94 (s, 3H); MS (ES+): m/z=196.1 (M−H)⁻; LCMS(Method B): T_(R=2.55) min.

Example 101

5-Methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde (98)

A mixture of 4-hydroxy-5-methoxy-2-nitrobenzaldehyde (97) (50.00 g,253.6 mmol), TIPS—Cl (59.70 mL, 279.0 mmol) and imidazole (51.80 g,760.9 mmol) was heated at 100° C. for 30 min. The reaction mixture waspoured into ice-cold water and extracted with ethyl acetate (3×500 mL).The organic layer was dried over Na₂SO₄, filtered and concentrated underreduced pressure. The resulting residue was purified by columnchromatography (silica), eluting with ethyl acetate/hexane (isocratic5%), to give the title compound (57.50 g, 64%) as a yellow solid.

¹H NMR (400 MHz, CDCl₃) δ 10.42 (s, 1H), 7.59 (s, 1H), 7.40 (s, 1H),3.95 (s, 3H), 1.33-1.24 (m, 3H), 1.07 (s, 18H).

Example 102

5-Methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic Acid (99)

A solution of sodium chlorite (45.97 g, 406.7 mmol, 80% technical grade)and NaH₂PO₄.2H₂O (35.53 g, 227.7 mmol) in water (200 mL) was added to asolution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde(98) (57.50 g, 162.7 mmol) in THF (800 mL) at room temperature. H₂O₂(30% w/w, 235 mL, 2.28 mol) was immediately added to the vigorouslystirred biphasic mixture. The starting material dissolved and thetemperature of the reaction mixture rose to 45° C. After 30 min, thereaction was judged to have completed by TLC. The mixture was acidifiedto pH=3-4 with citric acid. The mixture was extracted with ethyl acetate(3×500 mL). The combined extracts were washed with water (150 mL) andbrine (150 mL), dried over anhydrous Na₂SO₄, filtered and concentratedunder vacuum. The resulting residue was purified by columnchromatography (silica), eluting with ethyl acetate/hexane (isocratic10%) followed by methanol/dichloromethane (from 0% to 10%), to give thetitle compound (38.00 g, 63%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 7.35 (s, 1H), 7.25 (s, 1H), 3.91(s, 3H), 1.26 (q, J=7.4 Hz, 3H), 1.09 (d, J=7.4 Hz, 18H); MS (ES+):m/z=368.1 (M−H)⁻; LCMS (Method B): T_(R=4.75) min.

Example 103

(S)—(2-(Hydroxymethyl)piperidin-1-yl)(5-methoxy-2-nitro-4((triisopropylsilyl)oxy)phenyl)methanone(100)

A solution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid(99) (28.00 g, 75.8 mmol), HATU (31.70 g, 83.4 mmol) and dry Et₃N (44mL) in dry DCM (300 mL) was stirred at room temperature for 30 min.(S)—Piperidin-2-ylmethanol (11.35 g, 98.5 mmol) was added and thereaction mixture was stirred at room temperature for 2 h. The reactionmixture was partitioned between DCM (50 mL×2) and water (100 mL).Organic layer was dried over Na₂SO₄, filtered and concentrated underreduced pressure. The resulting residue was purified by columnchromatography (silica), eluting with ethyl acetate/hexane (from 50% to75%), to give the title compound (20.00 g, 57%) as a yellow solid.

¹H NMR (400 MHz, CDCl₃) δ 7.68-7.65 (m, 1H), 7.03-6.65 (m, 1H),5.04-4.69 (m, 1H), 4.12-4.05 (m, 0.41H), 4.01-3.95 (m, 0.46H), 3.92-3.89(m, 2.57H), 3.83-3.74 (m, 1.47H), 3.64-3.59 (m, 0.35H), 3.45-3.40 (m,0.28H), 3.21-3.01 (m, 1.39H), 2.87-2.79 (m, 0.38H), 1.97-1.94 (m,0.55H), 1.88-1.77 (m, 0.58H), 1.73-1.62 (m, 3H), 1.56-1.44 (m, 2H),1.29-1.24 (m, 3H), 1.09 (d, J=7.3 Hz, 18H); MS (ES+): m/z=467.3 (M+H)⁺;LCMS (Method A): T_(R=4.75) min.

Example 104

(S)—(2-Amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(hydroxymethyl)piperidin-1-yl)methanone(101)

A mixture of(S)—(2-(hydroxymethyl)piperidin-1-yl)(5-methoxy-2-nitro-4((triisopropylsilyl)oxy)phenyl)methanone(100) (10.0 g, 21.4 mmol), palladium on activated charcoal (10% wt.basis) (1.00 g) in methanol (100 mL) was stirred at room-temperatureunder H₂ for 18 h. The reaction mixture was filtered through Celite® andthe cake was washed with ethyl acetate (150 mL). The filtrate wasconcentrated under reduced pressure. The resulting residue was purifiedby column chromatography (silica), eluting with ethyl acetate/hexane(from 50% to 67%), to give the title compound (8.00 g, 85%) as a yellowoil.

¹H NMR (400 MHz, CDCl₃) δ 6.67 (s, 1H), 6.30 (s, 1H), 4.00-3.81 (m, 4H),3.72 (s, 3H), 3.57 (s, 1H), 3.08 (s, 1H), 1.68-1.64 (m, 4H), 1.57-1.43(m, 2H), 1.28-1.17 (m, 3H), 1.08 (d, J=7.4 Hz, 18H); MS (ES+): m/z=437.3(M+H)⁺; LCMS (Method B): t_(R=1.94) min.

Example 105

Allyl (S)—(2-(2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5((triisopropylsilyl)oxy)phenyl)carbamate (102)

To a stirred solution of(S)—(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(hydroxymethyl)piperidin-1-yl)methanone(101) (22.00 g, 50.4 mmol) and pyridine (7.97 g, 100.8 mmol) indichloromethane (300 mL) was added allyl chloroformate (6.38 g, 52.9mmol) dropwise at −10° C. After 30 min, the reaction was judged to havecompleted by TLC. Reaction mixture was diluted with dichloromethane (500mL) and washed with a saturated aqueous solution of copper (II) sulfate(150 mL), water (100 mL) and a saturated aqueous solution of sodiumhydrogen carbonate (100 mL). The organic layer was dried over Na₂SO₄,filtered and concentrated under reduced pressure. The resulting residuewas purified by column chromatography (silica), eluting with ethylacetate/hexane (from 50% to 75%), to give the title compound (17.00 g,65%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 7.62 (s, 1H), 6.75 (s, 1H), 5.92(ddt, J=17.2, 10.7, 5.5 Hz, 1H), 5.32 (dt, J=17.3, 1.7 Hz, 1H), 5.20(dt, J=10.6, 1.4 Hz, 1H), 4.61 (dt, J=5.5, 1.5 Hz, 2H), 3.88 (t, J=10.7Hz, 1H), 3.76 (s, 3H), 3.61-3.57 (m, 1H), 3.20-3.02 (m, 2H), 2.03 (s,1H), 1.65-1.62 (m, 3H), 1.53-1.40 (m, 2H), 1.29-1.24 (m, 4H), 1.11-1.08(m, 18H); MS (ES+): m/z=522.3 (M+H)⁺; LCMS (Method A): T_(R=5.23) min.

Example 106

Allyl(6aS)-6-hydroxy-2-methoxy-12-oxo-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(103)

A mixture of allyl(S)—(2-(2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5((triisopropylsilyl)oxy)phenyl)carbamate(102) (17.00 g, 32.7 mmol), TEMPO (510 mg, 3.3 mmol) and PIDA (12.62 g,39.2 mmol) in DCM (150 mL) was stirred at room-temperature for 16 h. Thereaction mixture was diluted with DCM (350 mL), washed with aq. Na₂SO₃(100 mL), aq. NaHCO₃ (100 mL) and brine (100 mL). The organic phase wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. The resulting residue was purified by column chromatography(silica), eluting with ethyl acetate/hexane (from 50% to 75%), to givethe title compound (13.00 g, 77%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 7.13 (s, 1H), 6.65 (s, 1H), 5.90 (d, J=10.3Hz, 1H), 5.78 (td, J=10.6, 5.3 Hz, 1H), 5.19-5.13 (m, 2H), 4.60 (dd,J=13.1, 5.8 Hz, 1H), 4.52-4.40 (m, 1H), 4.35 (dt, J=13.6, 4.5 Hz, 1H),3.84 (s, 3H), 3.57-3.39 (m, 2H), 3.14-2.99 (m, 1H), 2.08-1.99 (m, 1H),1.76-1.61 (m, 5H), 1.25-1.18 (m, 3H), 1.09-1.05 (m, 18H); MS (ES+):m/z=519.3 (M+H)⁺; LCMS (Method A): T_(R=2.41) min.

Example 107

Allyl(6aS)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(104)

A mixture of allyl(6aS)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(103) (14.00 g, 27.0 mmol), DHP (22.70 g, 269.9 mmol) and pTSA.H₂O (140mg, 0.76 mmol) in THF (130 mL) was stirred at room-temperature for 18 h.The reaction mixture was diluted with ethyl acetate (360 mL), washedwith aq. NaHCO₃ (200 mL) and brine (100 mL). The organic phase was driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressure.The resulting residue was purified by column chromatography (silica),eluting with ethyl acetate/hexane (isocratic 17%), to give the titlecompound (12.50 g, 77%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 7.13 (s, 0.38H), 7.10 (s, 0.53H), 6.90 (s,0.50H), 6.52 (s, 0.35H), 6.15 (d, J=10.0 Hz, 0.37H), 5.98 (d, J=10.0 Hz,0.51H), 5.80-5.68 (m, 0.88H), 5.17-4.94 (m, 2.73H), 4.64-4.21 (m, 3H),3.91-3.85 (m, 0.85H), 3.83 (d, J=1.8 Hz, 3H), 3.66-3.39 (m, 2H),3.14-3.00 (m, 1H), 2.08-1.87 (m, 1H), 1.83-1.33 (m, 12H), 1.26-1.19 (m,3H), 1.08-1.05 (m, 18H); MS (ES+): m/z=603.4 (M+H)⁺; LCMS (Method A):T_(R=6.41) min.

Example 108

Allyl(6aS)-3,6-dihydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(105)

A 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (0.3mL) was added to a mixture of allyl(6aS)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(103) (50 mg, 0.10 mmol) in 1,4-dioxane (2 mL). the reaction mixture wasstirred for 30 min and it was then washed with a saturated aqueoussolution of sodium chloride (30 mL). The aqueous solution was washedwith ethyl acetate (2×30 mL) and the organic solvent was concentratedunder vacuum. The resulting residue was purified by columnchromatography (silica), eluting with acetone/dichloromethane to givethe title compound (36 mg, 99%) as a yellow oil.

MS (ES+): m/z=363 (M+H)⁺; LCMS (Method B): T_(R=2.60) min.

Example 109

(S)—3-Hydroxy-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one(106)

To a solution of allyl(6aS)-3,6-dihydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(105) (36.0 mg, 0.10 mmol) in dichloromethane (3 mL) was sequentiallyadded tetrakis(triphenylphosphine)palladium(0) (5.7 mg, 5 mol %), andpyrrolidine (9.7 mL, 0.12 mmol). The reaction mixture was stirred atroom temperature for 30 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%) to give the title compound (20 mg, 76%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 7.89 (d, J=5.9 Hz, 1H), 7.41 (s, 1H), 6.84 (s,1H), 3.92 (s, 3H), 3.80-3.69 (m, 2H), 3.28-3.16 (m, 1H), 2.18-2.01 (m,2H), 1.99-1.81 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 163.2, 148.4, 145.9,145.6, 120.8, 112.0, 111.2, 56.2, 49.6, 39.7, 24.4, 22.9, 18.3; MS(ES+): m/z=279 (M+H+H₂O)⁺; LCMS (Method A): T_(R=4.68) min.

Example 110

(S)—4-((2-Methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicAcid (107)

To a solution4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (50.0 mg, 0.09 mmol) in dichloromethane (3 mL) was sequentiallyadded tetrakis(triphenylphosphine)palladium(0) (5.4 mg, 5 mol %), andpyrrolidine (9.0 μL, 0.11 mmol). The reaction mixture was stirred atroom temperature for 30 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%) to give the title compound (25 mg, 80%) as a cream solid.

¹H NMR (400 MHz, CDCl₃) δ 7.87 (br. s., 1H), 7.39 (s, 1H), 6.81 (s, 1H),4.16-4.00 (m, 3H), 3.88 (s, 3H), 3.83-3.72 (m, 2H), 3.29-3.15 (m, 1H),2.52 (br. s., 2H), 2.20-1.77 (m, 8H); ¹³C NMR (100 MHz, CDCl₃) δ 176.5,167.7, 150.7, 139.2, 128.5, 121.1, 111.7, 109.7, 67.8, 56.1, 49.7, 39.8,30.3, 24.4, 22.9, 18.2; MS (ES+): m/z=347 (M+H)⁺; LCMS (Method B):T_(R=2.73) min.

Example 111

Allyl(6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(108)

To a solution of allyl(6aS)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-3-((triisopropylsilyl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(103) (2.0 g, 3.32 mmol) in THF (10 mL) was added TBAF (1 M, 5 mL). Thereaction mixture was stirred at room temperature for 5 min andconcentrated under reduced pressure. The resulting residue was purifiedby column chromatography (silica), eluting with ethyl acetate/hexane(from 0% to 100%), to give the title compound (1.2 g, 83%) as a whitesolid.

MS (ES+): m/z=446.7 (M+H)⁺; LCMS (Method A): T_(R=3.22) min.

Example 112

Allyl(6aS)-2-methoxy-3-((3-(2-methoxy-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(109)

A mixture of allyl(6aS)-3-hydroxy-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(108) (1.2 g, 2.69 mmol), methyl 2-(3-(bromomethyl)phenyl)acetate (686mg, 2.82 mmol) and K₂CO₃ (560 mg, 4.05 mmol), in DMF (15 mL) was stirredat room temperature for 18 h. The reaction mixture was poured intoice-cold water (50 mL) and extracted with ethyl acetate (2×80 mL). Thecombined organic extracts were concentrated in vacuo. The resultingresidue was purified by column chromatography (silica), eluting withethyl acetate/hexane (from 0% to 100%), to give the title compound (1.08g, 66%) as a colourless oil.

MS (ES+): m/z=608.8 (M+H)⁺; LCMS (Method B): T_(R=3.73) min.

Example 113

2-(3-((((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)aceticAcid (110)

To a solution of(6aS)-2-methoxy-3-((3-(2-methoxy-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(109) (1.08 g, 1.77 mmol) in 1,4-dioxane (9 mL) was added an aqueoussolution of sodium hydroxide (1 M, 9 mL, 9 mmol). The reaction mixturewas stirred at room temperature for 1 h and was then concentrated invacuo, after which water (15 mL) was added and the aqueous layer wasacidified to pH=4 with an aqueous solution of citric acid (1 M, 10 mL).The aqueous layer was then extracted with ethyl acetate (2×50 mL). Theorganic layer was washed with brine (50 mL). The combined organicextracts were dried over sodium sulfate, filtered and concentrated togive the title compound (900 mg, 86%) as a white solid. The product wascarried through to the next step without any further purification.

MS (ES+): m/z=594.7 (M+H)⁺; LCMS (Method B): T_(R=3.43) min.

Example 114

Allyl(6aS)-2-methoxy-3-((3-(2-((5-(methoxycarbonyl)-1-methy-1H-pyrrol-3-yl)amino)-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(111)

A solution of2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)aceticacid (110) (450 mg, 0.757 mmol) in N,N—dimethylformamide (4 mL) wascharged with N,N—dimethylpyridin-4-amine (232 mg, 1.90 mmol) andN—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (291 mg,1.52 mmol) and it was stirred for 30 min at room temperature. Methyl4-amino-1-methyl-1H-pyrrole-2-carboxylate hydrochloride (145 mg, 0.761mmol) was added and the solution was stirred for further 18 h. Thereaction mixture was poured into ice-cold water (50 mL) and extractedwith ethyl acetate (2×80 mL). The combined organic extracts wereconcentrated in vacuo. The resulting residue was purified by columnchromatography (silica), eluting with ethyl acetate/hexane (from 0% to100%), to give the title compound (245 mg, 44%) as a brown oil.

MS (ES+): m/z=730.8 (M+H)⁺; LCMS (Method B): T_(R=3.58) min.

Example 115

Methyl(S)—4-(2-(3-(((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-pyrrole-2-carboxylate(112)

To a solution of allyl(6aS)-2-methoxy-3-((3-(2-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(111) (20 mg, 0.0274 mmol) in dichloromethane (1 mL) was sequentiallyadded tetrakis(triphenylphosphine)palladium(0) (1.6 mg, 5 mol %), andpyrrolidine (2.7 μL, 0.0329 mmol). The reaction mixture was stirred atroom temperature for 30 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%) to give the title compound (11.0 mg, 74%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, J=5.9 Hz, 1H), 7.46 (s, 1H),7.34-7.40 (m, 5H), 6.86 (s, 1H), 6.62 (d, J=1.9 Hz, 1H), 5.18 (q, J=12.5Hz, 2H), 4.24 (d, J=13.7 Hz, 1H), 3.94 (s, 3H), 3.88 (s, 1H), 3.86 (s,3H), 3.78 (s, 3H), 3.73 (s, 1H), 3.71 (s, 1H), 3.69 (s, 2H), 3.18-3.27(m, 1H), 1.80-1.91 (m, 3H), 1.70-1.77 (m, 2H); MS (ES+): m/z=544.7(M+H)⁺; LCMS (Method B): T_(R=3.03) min; LCMS (Method A): T_(R=6.35)min.

Example 116

Allyl(6aS)-3-((3-(2-((2-(ethoxycarbonyl)-1-methyl-1-imidazol-4-yl)amino)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(113)

A solution of2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,1,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)aceticacid (110) (450 mg, 0.757 mmol) in N,N—dimethylformamide (4 mL) wascharged with N,N—dimethylpyridin-4-amine (232 mg, 1.90 mmol) andN—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (291 mg,1.52 mmol) and it was stirred for 30 min at room temperature. Ethyl4-amino-1-methyl-1H-imidazole-2-carboxylate hydrochloride (156 mg, 0.759mmol) was added and the solution was stirred for further 18 h. Thereaction mixture was poured into ice-cold water (50 mL) and extractedwith ethyl acetate (2×80 mL). The combined organic extracts wereconcentrated in vacuo. The resulting residue was purified by columnchromatography (silica), eluting with ethyl acetate/hexane (from 0% to100%), followed by methanol/dichloromethane (from 0% to 100%), to givethe title compound (407 mg, 72%) as a colourless oil.

MS (ES+): m/z=745.9 (M+H)⁺; LCMS (Method B): T_(R=3.55) min.

Example 117

Ethyl(S)—4-(2-(3-(((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-imidazole-2-carboxylate(114)

To a solution of allyl(6aS)-3-((3-(2-((2-(ethoxycarbonyl)-1-methyl-1H-imidazol-4-yl)amino)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(113) (20 mg, 0.0268 mmol) in dichloromethane (1 mL) was sequentiallyadded tetrakis(triphenylphosphine)palladium(0) (1.6 mg, 5 mol %), andpyrrolidine (2.6 mL, 0.0268 mmol). The reaction mixture was stirred atroom temperature for 30 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 5%) to give the title compound (4.05 mg, 29%) as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 7.89 (br. s., 1H), 7.54-7.51 (m, 1H), 7.46(br. s., 1H), 7.43-7.32 (m, 3H), 7.26-7.21 (m, 1H), 6.83 (br. s., 1H),5.25-5.08 (m, 2H), 4.42-4.35 (m, 2H), 4.24 (d, J=14.1 Hz, 1H), 4.00-3.97(m, 2H), 3.97-3.94 (m, 3H), 3.77-3.71 (m, 3H), 3.32-3.12 (m, 1H),2.16-2.01 (m, 2H), 1.96 (br. s., 1H), 1.89-1.64 (m, 5H), 1.42-1.36 (m,3H); MS (ES+): m/z=559.9 (M+H)⁺; LCMS (Method B): T_(R=2.95) min; LCMS(Method A): T_(R=6.15) min.

Example 118

4-(2-(3-((((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-imidazole-2-carboxylicAcid (115)

To a solution of allyl(6aS)-3-((3-(2-((2-(ethoxycarbonyl)-1-methyl-1H-imidazol-4-yl)amino)-2-oxoethyl)benzyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(114) (330 mg, 0.442 mmol) in 1,4-dioxane (2.5 mL) was added an aqueoussolution of sodium hydroxide (1 M, 2.5 mL, 2.5 mmol). The reactionmixture was stirred at room temperature for 1.5 h and was thenconcentrated in vacuo, after which water (15 mL) was added and theaqueous layer was acidified to pH=4 with an aqueous solution of citricacid (1 M, 10 mL). The aqueous layer was then extracted with ethylacetate (2×50 mL). The organic layer was washed with brine (50 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated in vacuo The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 10%) to give the title compound (133 mg, 42%) as a cream solid.

MS (ES+): m/z=717.8 (M+H)⁺; LCMS (Method B): T_(R=3.23) min.

Example 119

Methyl2-(4-(2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[d]thiazole-5-carboxylate(116)

A solution of4-(2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-imidazole-2-carboxylicacid (115) (65 mg, 0.0906 mmol) in anhydrous dichloromethane (1 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (37 mg, 0.0973 mmol) and anhydroustriethylamine (54 μL, 0.387 mmol). The reaction mixture was stirred atroom temperature for 20 min. Methyl2-aminobenzo[d]thiazole-5-carboxylate (19 mg, 0.0912 mmol) was thenadded and the resulting mixture was stirred at room temperature for 16h. The reaction mixture was quenched with a saturated aqueous solutionof sodium hydrogen carbonate (20 mL) and extracted with dichloromethane(2×50 mL). The combined organic extracts were washed with watercontaining a few drops of acetic acid (30 mL). The organic layer wasthen dried over sodium sulfate, filtered and concentrated in vacuo. Theresulting residue was then purified by column chromatography (silica),eluting with ethyl acetate/hexane (from 0% to 100%), followed bymethanol/dichloromethane (from 0% to 100%), to give the title compound(23 mg, 28%) as a yellow oil.

MS (ES+): m/z=907.8 (M+H)⁺; LCMS (Method B): T_(R=3.98) min.

Example 120

2-(4-(2-(3-((((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[d]thiazole-5-carboxylicAcid (117)

To a solution of methyl2-(4-(2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[d]thiazole-5-carboxylate(116) (10 mg, 0.011 mmol) in 1,4-dioxane (0.5 mL) was added an aqueoussolution of sodium hydroxide (1 M, 0.5 mL, 0.5 mmol). The reactionmixture was stirred at room temperature for 1 h and was thenconcentrated in vacuo, after which water (15 mL) was added and theaqueous layer was acidified to pH=4 with an aqueous solution of citricacid (1 M, 10 mL). The aqueous layer was then extracted with ethylacetate (2×50 mL). The organic layer was washed with brine (50 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated in vacuo to give the title compound (9.7 mg, 98%) as awhite solid. The product was carried through to the next step withoutany further purification

MS (ES+): m/z=893.8 (M+H)⁺; LCMS (Method B): T_(R=3.58) min.

Example 121

Allyl(6aS)-2-methoxy-3-((3-(2-((1-methyl-2-((5-(phenylcarbamoyl)benzo[d]thiazol-2-yl)carbamoyl)-1H-imidazol-4-yl)amino)-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(118)

A solution of2-(4-(2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[d]thiazole-5-carboxylicacid (117) (9.7 mg, 0.0109 mmol) in anhydrous dichloromethane (0.5 mL)was charged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (4.3 mg, 0.0113 mmol) and anhydroustriethylamine (6.5 μL, 0.0466 mmol). The reaction mixture was stirred atroom temperature for 20 min. Aniline (1.0 μL, 0.0109 mmol) was thenadded and the resulting mixture was stirred at room temperature for 2 h.The reaction mixture was quenched with a saturated aqueous solution ofsodium hydrogen carbonate (20 mL) and extracted with dichloromethane(2×50 mL). The combined organic extracts were washed with watercontaining a few drops of acetic acid (30 mL). The organic layer wasthen dried over sodium sulfate, filtered and concentrated in vacuo. Theresulting residue was then purified by column chromatography (silica),eluting with ethyl acetate/hexane (from 0% to 100%), followed bymethanol/dichloromethane (from 0% to 100%), to give the title compound(3.4 mg, 32%) as a cream film.

MS (ES+): m/z=968.9 (M+H)⁺; LCMS (Method B): T_(R=3.88) min.

Example 122

(S)—2-(4-(2-(3-(((2-Methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-1H-imidazole-2-carboxamido)-N—phenylbenzo[d]thiazole-5-carboxamide(119)

To a solution of allyl(6aS)-2-methoxy-3-((3-(2-((1-methyl-2-((5-(phenylcarbamoyl)benzo[d]thiazol-2-yl)carbamoyl)-1H-imidazol-4-yl)amino)-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(118) (3.4 mg, 0.00351 mmol) in dichloromethane (0.5 mL) wassequentially added tetrakis(triphenylphosphine)palladium(0) (0.2 mg, 5mol %), and pyrrolidine (0.4 μL, 0.00487 mmol). The reaction mixture wasstirred at room temperature for 1 h. The reaction mixture concentratedin vacuo and subjected to high vacuum for 40 min until excesspyrrolidine was removed. The resulting residue was then purified bycolumn chromatography (silica), eluting with methanol/dichloromethane(from 0% to 100%) to give the title compound (1.5 mg, 55%) as a creamfilm.

¹H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 10.33 (s, 1H), 8.36 (br. s.,1H), 8.10 (br. s., 1H), 7.88 (d, J=7.0 Hz, 1H), 7.83 (d, J=8.2 Hz, 1H),7.58 (br. s., 1H), 7.45-7.27 (m, 6H), 7.15-7.07 (m, 1H), 5.22-4.97 (m,2H), 4.01 (s, 2H), 3.83-3.79 (m, 1H), 3.71 (br. s., 1H), 3.70 (s, 2H),3.66-3.64 (m, 2H), 3.63-3.56 (m, 1H), 3.46-3.37 (m, 1H), 2.11-1.95 (m,1H), 1.90-1.82 (m, 1H), 1.80-1.64 (m, 2H), 1.62-1.45 (m, 2H), 1.18-1.09(m, 2H); MS (ES+): m/z=782.7 (M+H)⁺; LCMS (Method B): T_(R=3.38) min;LCMS (Method A): T_(R=7.28) min.

Example 123

Allyl(6aS)-3-(4-((2-(ethoxycarbonyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(120)

A solution of4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (9) (500 mg, 0.939 mmol) in N,N—dimethylformamide (5 mL) wascharged with N,N—dimethylpyridin-4-amine (287 mg, 2.35 mmol) andN—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (360 mg,1.88 mmol) and it was stirred for 15 min at room temperature. Ethyl4-amino-1-methyl-1H-imidazole-2-carboxylate hydrochloride (195 mg, 0.948mmol) was added and the solution was stirred for further 18 h. Thereaction mixture was poured into ice-cold water (50 mL) and extractedwith ethyl acetate (2×80 mL). The combined organic extracts wereconcentrated in vacuo to give the title compound (642 mg, 99%) as abrown oil. The product was carried through to the next step without anyfurther purification.

MS (ES+): m/z=683.9 (M+H)⁺; LCMS (Method B): T_(R=3.35) min.

Example 124

4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxylicAcid (121)

To a solution of allyl(6aS)-3-(4-((2-(ethoxycarbonyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(120) (642 mg, 0.939 mmol) in 1,4-dioxane (5 mL) was added an aqueoussolution of sodium hydroxide (1 M, 5 mL, 5 mmol). The reaction mixturewas stirred at room temperature for 1.5 h and was then concentrated invacuo, after which water (15 mL) was added and the aqueous layer wasacidified to pH=4 with an aqueous solution of citric acid (1 M, 10 mL).The aqueous layer was then extracted with ethyl acetate (2×50 mL). Theorganic layer was washed with brine (50 mL). The combined organicextracts were dried over sodium sulfate, filtered and concentrated invacuo to give the title compound (615 mg, 99%) as a cream solid. Theproduct was carried through to the next step without any furtherpurification

MS (ES+): m/z=655.8 (M+H)⁺; LCMS (Method B): T_(R=3.00) min.

Example 125

Methyl2-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-4-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[d]thiazole-5-carboxylate(122)

A solution of4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxylicacid (121) (100 mg, 0.153 mmol) in N,N—dimethylformamide (1 mL) wascharged with N,N—dimethylpyridin-4-amine (47 mg, 0.385 mmol) andN—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (59 mg,0.308 mmol) and it was stirred for 30 min at room temperature. Methyl2-aminobenzo[d]thiazole-5-carboxylate (32 mg, 0.154 mmol) was added andthe solution was stirred for further 18 h. The reaction mixture waspoured into ice-cold water (50 mL) and extracted with ethyl acetate(2×80 mL). The combined organic extracts were concentrated in vacuo. Theresulting residue was purified by column chromatography (silica),eluting with ethyl acetate/hexane (from 0% to 100%), followed bymethanol/dichloromethane (from 0% to 100%), to give the title compound(7.0 mg, 5%) as a yellow solid.

MS (ES+): m/z=845.8 (M+H)⁺; LCMS (Method B): T_(R=3.80) min.

Example 126

2-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[d]thiazole-5-carboxylicAcid (123)

To a solution of methyl2-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[d]thiazole-5-carboxylate(122) (7 mg, 0.00828 mmol) in 1,4-dioxane (0.5 mL) was added an aqueoussolution of sodium hydroxide (1 M, 0.5 mL, 0.5 mmol). The reactionmixture was stirred at room temperature for 1 h and was thenconcentrated in vacuo, after which water (15 mL) was added and theaqueous layer was acidified to pH=4 with an aqueous solution of citricacid (1 M, 10 mL). The aqueous layer was then extracted with ethylacetate (2×50 mL). The organic layer was washed with brine (50 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated in vacuo to give the title compound (6.4 mg, 93%) as awhite solid. The product was carried through to the next step withoutany further purification

MS (ES+): m/z=829.8 (M−H)⁻; LCMS (Method B): T_(R=3.47) min.

Example 127

Allyl(6aS)-2-methoxy-3-(4-((1-methyl-2-((5-(phenylcarbamoyl)benzo[d]thiazol-2-yl)carbamoyl)-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(124)

A solution of2-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[d]thiazole-5-carboxylicacid (123) (6.4 mg, 0.00769 mmol) in anhydrous dichloromethane (0.5 mL)was charged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (3.0 mg, 0.00789 mmol) and anhydroustriethylamine (5.0 μL, 0.0359 mmol). The reaction mixture was stirred atroom temperature for 20 min. Aniline (1.0 μL, 0.0109 mmol) was thenadded and the resulting mixture was stirred at room temperature for 2 h.The reaction mixture was quenched with a saturated aqueous solution ofsodium hydrogen carbonate (20 mL) and extracted with dichloromethane(2×50 mL). The combined organic extracts were washed with watercontaining a few drops of acetic acid (30 mL). The organic layer wasthen dried over sodium sulfate, filtered and concentrated in vacuo. Theresulting residue was then purified by column chromatography (silica),eluting with ethyl acetate/hexane (from 0% to 100%), followed bymethanol/ethyl acetate (from 0% to 100%), to give the title compound(2.2 mg, 32%) as a cream film.

MS (ES+): m/z=906.8 (M+H)⁺; LCMS (Method B): T_(R=3.77) min.

Example 128

(S)—2-(4-(4-((2-Methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)-N—phenylbenzo[d]thiazole-5-carboxamide(125)

To a solution allyl(6aS)-2-methoxy-3-(4-((1-methyl-2-((5-(phenylcarbamoyl)benzo[d]thiazol-2-yl)carbamoyl)-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(124) (2.2 mg, 0.00243 mmol) in dichloromethane (0.5 mL) wassequentially added tetrakis(triphenylphosphine)palladium(0) (0.2 mg, 7mol %), and pyrrolidine (0.3 μL, 0.00365 mmol). The reaction mixture wasstirred at room temperature for 1 h. The reaction mixture concentratedin vacuo and subjected to high vacuum for 40 min until excesspyrrolidine was removed. The resulting residue was then purified bycolumn chromatography (silica), eluting with methanol/dichloromethane(from 0% to 100%) to give the title compound (1.7 mg, 97%) as a creamfilm.

¹H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 10.32 (br. s., 2H), 8.35 (br.s., 1H), 8.00 (d, J=5.9 Hz, 1H), 7.85-7.80 (m, 2H), 7.73 (br. s., 1H),7.68-7.66 (m, 2H), 7.59 (br. s., 1H), 7.36 (t, J=7.8 Hz, 2H), 7.23 (s,1H), 7.12 (d, J=7.0 Hz, 1H), 4.12 (s, 3H), 4.04-3.99 (m, 3H), 3.85-3.78(m, 2H), 3.71 (s, 1H), 3.61-3.56 (m, 1H), 3.08 (s, 1H), 2.68-2.65 (m,2H), 2.08-1.96 (m, 4H), 1.65-1.60 (m, 4H); MS (ES+): m/z=720.9 (M+H)⁺;LCMS (Method B): T_(R=3.25) min; LCMS (Method A): T_(R=6.98) min.

Example 129

4-(4-(4-((tert-Butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicAcid (126)

To a solution of methyl4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate(57) (3.1 g, 6.85 mmol) in 1,4-dioxane (120 mL) was added an aqueoussolution of sodium hydroxide (1 M, 120 mL, 120 mmol). The reactionmixture was stirred at room temperature for 18 h and was thenconcentrated in vacuo, after which water (80 mL) was added and theaqueous layer was acidified to pH=4 with an aqueous solution of citricacid (1 M, 80 mL). The aqueous layer was then extracted with ethylacetate (2×150 mL). The organic layer was washed with brine (150 mL).The combined organic extracts were dried over sodium sulfate, filteredand concentrated in vacuo to give the title compound (2.5 g, 83%) as acream solid. The product was carried through to the next step withoutany further purification.

MS (ES+): m/z=438.8 (M+H)⁺; LCMS (Method B): T_(R=3.27) min.

Example 130

tert-Butyl(1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)carbamate(127)

A solution of4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (126) (2.0 g, 4.56 mmol) in anhydrous dichloromethane (14 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (1.85 g, 4.87 mmol) and anhydroustriethylamine (2.7 mL, 19.4 mmol). The reaction mixture was stirred atroom temperature for 30 min. Aniline (440 mL, 4.82 mmol) was added andthe resulting mixture was stirred at room temperature for 16 h. Thereaction mixture was quenched with a saturated aqueous solution ofsodium hydrogen carbonate (50 mL) and extracted with dichloromethane(2×150 mL). The combined organic extracts were washed with watercontaining a few drops of acetic acid (75 mL). The organic layer wasthen dried over sodium sulfate, filtered and concentrated in vacuo, togive the title compound (2.0 g, 85%) as a cream film.

¹H NMR (400 MHz, DMSO-d6) δ 9.83 (br. s., 1H), 9.77 (br. s., 1H), 9.12(br. s., 1H), 7.73 (br. s., 4H), 7.50 (d, J=7.4 Hz, 2H), 7.46 (br. s.,1H), 7.41 (br. s., 1H), 7.33 (br. s., 2H), 7.06 (br. s., 1H), 6.94 (br.s., 2H), 3.91 (br. s., 3H), 3.81 (br. s., 3H), 1.46 (br. s., 9H) MS(ES+): m/z=513.8 (M+H)⁺; LCMS (Method B): T_(R=3.70) min.

Example 131

4-Amino-1-methyl-N—(4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)-1H-pyrrole-2-carboxamideHydrochloride (128)

tert-Butyl(1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)carbamate(127) (1.0 g, 1.95 mmol) was dissolved in hydrochloric acid (4 M in1,4-dioxane) (5 mL) and the reaction mixture was stirred at roomtemperature for 1 h. The reaction mixture was concentrated in vacuo togive the title compound (975 mg, 99%) as a brown solid. The product wascarried through to the next step without any further purification.

MS (ES+): m/z=413.8 (M+H)⁺; LCMS (Method B): T_(R=3.07) min.

Example 132

Allyl(6aS)-2-methoxy-3-((3-(2-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(129)

A solution of2-(3-((((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)aceticacid (110) (50 mg, 0.084 mmol) in N,N—dimethylformamide (1 mL) wascharged with N,N—dimethylpyridin-4-amine (30 mg, 0.245 mmol) andN—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (32 mg,0.168 mmol) and it was stirred for 30 min at room temperature.4-Amino-1-methyl-N—(4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)-1H-pyrrole-2-carboxamidehydrochloride (128) (41.6 mg, 0.092 mmol) was added and the solution wasstirred for further 18 h. The reaction mixture was poured into ice-coldwater (50 mL) and extracted with ethyl acetate (2×80 mL). The combinedorganic extracts were concentrated in vacuo. The resulting residue waspurified by column chromatography (silica), eluting with ethylacetate/hexane (from 0% to 100%), to give the title compound (21 mg,25%) as a white solid.

MS (ES+): m/z=989.8 (M+H)⁺; LCMS (Method B): T_(R=4.35) min.

Example 133

(S)—4-(2-(3-(((2-Methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)methyl)phenyl)acetamido)-1-methyl-N—(4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)-1H-pyrrole-2-carboxamide(130)

To a solution allyl(6aS)-2-methoxy-3-((3-(2-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-2-oxoethyl)benzyl)oxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(129) (21 mg, 0.0212 mmol) in dichloromethane (0.5 mL) was sequentiallyadded tetrakis(triphenylphosphine)palladium(0) (1.2 mg, 5 mol %), andpyrrolidine (2.1 μL, 0.0256 mmol). The reaction mixture was stirred atroom temperature for 1 h. The reaction mixture concentrated in vacuo andsubjected to high vacuum for 40 min until excess pyrrolidine wasremoved. The resulting residue was then purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 100%) to give the title compound (8.0 mg, 47%) as a cream solid.

¹H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.83 (s, 1H), 9.80 (s, 1H),7.75 (s, 1H), 7.72 (d, J=2.3 Hz, 2H), 7.70 (s, 1H), 7.50 (d, J=8.6 Hz,2H), 7.46 (s, 1H), 7.44-7.39 (m, 2H), 7.35 (s, 2H), 7.33 (s, 2H), 7.31(br. s., 1H), 7.21 (S, 1H), 7.11-7.07 (m, 1H), 7.07-7.03 (m, 1H), 6.99(s, 1H), 6.66 (s, 1H), 6.12 (s, 1H), 5.15-4.95 (m, 2H), 3.91 (s, 3H),3.83 (s, 3H), 3.72-3.66 (m, 3H), 3.61 (s, 2H), 3.48-3.39 (m, 1H),1.93-1.71 (m, 2H), 1.69-1.47 (m, 5H), 1.25-1.22 (m, 1H); MS (ES+):m/z=804.0 (M+H)⁺; LCMS (Method B): T_(R=3.45) min; LCMS (Method A):T_(R=7.52) min.

Example 134

(S)—(2-(((tert-Butyldimethylsilyl)oxy)methyl)piperidin-1-yl)(5-methoxy-2-nitro-4((triisopropylsilyl)oxy)phenyl)methanone (131)

TBS-OTf (3.12 g, 11.8 mmol) was added to a solution of(S)—(2-(hydroxymethyl)piperidin-1-yl)(5-methoxy-2-nitro-4((triisopropylsilyl)oxy)phenyl)methanone(100) (5.50 g, 11.8 mmol) and 2,6-lutidine (5.05 g, 47.2 mmol) in dryDCM (50 mL) at 0° C. for 4 h. The reaction mixture was diluted with DCM(300 mL) and sequentially washed with water (50 mL), sat. aq. NaHCO₃ (50mL) and brine (50 mL). Organic layer was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The resulting residue was thenpurified by column chromatography (silica), eluting with ethylacetate/hexane (from 9% to 50%) to give the title compound (4.1 g, 60%)as a yellow oil.

¹H NMR (400 MHz, CDCl₃) δ 7.68 (s, 1H), 6.74-6.64 (m, 1H), 4.99-4.65 (m,1H), 3.91-3.86 (m, 3H), 3.81-3.61 (m, 1H), 3.57-3.39 (m, 0.62H),3.16-3.03 (m, 1H), 2.77 (d, J=12.4 Hz, 0.34H), 2.14 (d, J=13.4 Hz,00.31H), 1.86-1.44 (m, 5H), 1.31-1.25 (m, 3.75H), 1.10-1.04 (m, 18H),0.92-0.82 (m, 9H), 0.12-−0.04 (m, 6H);

MS (ES+): m/z=580.9 (M+H)⁺; LCMS (Method A): T_(R=9.21) min.

Example 135

(S)—(4-Hydroxy-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)piperidin-1-yl)methanone(132)

To a solution of(S)—(2-(((tert-butyldimethylsilyl)oxy)methyl)piperidin-1-yl)(5-methoxy-2-nitro-4((triisopropylsilyl)oxy)phenyl)methanone (131) (3.9 g, 6.72 mmol) in THF(40 mL) was added TBAF (1 M, 15 mL). The reaction mixture was stirred atroom temperature for 20 h and concentrated under reduced pressure togive the title compound (2.1 g, 99%) as a yellow oil. The product wascarried through to the next step without any further purification.

MS (ES+): m/z=310.8 (M+H)⁺; LCMS (Method B): T_(R=2.57) min.

Example 136

Methyl(S)—4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)butanoate(133)

A mixture of(S)—(4-hydroxy-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)piperidin-1-yl)methanone(132) (2.1 g, 6.77 mmol), methyl 4-bromobutanoate (900 μL, 7.13 mmol)and potassium carbonate (1.4 g, 10.13 mmol) in N,N—dimethylformamide (30mL) was stirred at room temperature for 18 h. The reaction mixture wasdiluted with water (60 mL) and extracted with ethyl acetate (2×100 mL).The combined organic extracts were concentrated in vacuo. The resultingresidue was purified by column chromatography (silica), eluting withmethanol/dichloromethane (from 0% to 100%), to give the title compound(2.3 g, 83%) as a yellow oil.

MS (ES+): m/z=410.8 (M+H)⁺; LCMS (Method B): T_(R=2.93) min.

Example 137

Methyl(S)—4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate(134)

Ammonium formate (2.95 g, 46.78 mmol) and palladium on activatedcharcoal (10% wt. basis) (5.0 g) were added to a solution of methyl(S)—4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)butanoate(133) in THF (90 mL) and water (10 mL). The reaction mixture was stirredat 35° C. for 2 h. The reaction mixture was filtered through Celite® andwashed with ethyl acetate (200 mL). The filtrate was concentrated invacuo to give the title compound (1.75 g, 79%) as an amber oil. Theproduct was carried through to the next step without any furtherpurification.

¹H NMR (400 MHz, CDCl₃) δ 6.68 (s, 1H), 6.24 (s, 1H), 4.53 (br. s., 1H),3.96 (t, J=6.2 Hz, 2H), 3.93-3.82 (m, 4H), 3.73 (s, 3H), 3.65 (d, J=0.8Hz, 3H), 3.55 (d, J=5.1 Hz, 1H), 3.06 (br. s., 1H), 2.49 (t, J=7.2 Hz,2H), 2.14-2.05 (m, 2H), 1.68-1.58 (m, 4H), 1.56-1.41 (m, 2H), 1.04-0.98(m, 1H); MS (ES+): m/z=380.8 (M+H)⁺; LCMS (Method B): T_(R=2.58) min.

Example 138

Methyl4-(5-((((4-((S)—2-((S)—2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)—2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate(135)

A solution of methyl(S)—4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate(134) (1.75 g, 4.60 mmol) and allyl((S)—3-methyl-1-(((S)—1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate(2.5 g, 4.61 mmol) in DMF (9 mL) was stirred at room temperature for 5min. 1H-Benzo[d][1,2,3]triazol-1-ol (622 mg, 4.60 mmol) was added to thereaction mixture which was heated to 60° C. for 22 h. The reactionmixture was partitioned between ethyl acetate (2×50 mL) and water (30mL). The combined organic extracts were concentrated in vacuo. Theresulting residue was purified by column chromatography (silica),eluting with methanol/dichloromethane (from 0% to 100%), to give thetitle compound (2.5 g, 62%) as a beige solid.

MS (ES+): m/z=783.9 (M+H)⁺; LCMS (Method B): T_(R=3.15) min.

Example 139

4-(5-((((4-((S)—2-((S)—2-(((Allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)—2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoicAcid (136)

To a solution of methyl4-(5-((((4-((S)—2-((S)—2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)—2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate(135) (2.5 g, 2.87 mmol) in 1,4-dioxane (12 mL) was added an aqueoussolution of sodium hydroxide (0.5 M, 18 mL, 9.00 mmol) dropwise. Thereaction mixture was stirred at room temperature for 4 h. The reactionmixture was acidified to pH=1 with 1 M HCl (10 mL) before being dilutedwith water (30 mL) and extracted with ethyl acetate (2×100 mL). Thecombined organic extracts were concentrated in vacuo. The resultingresidue was purified by column chromatography (silica), eluting withmethanol/dichloromethane (from 0% to 100%), to give the title compound(1.2 g, 54%) as a cream solid.

MS (ES+): m/z=769.8 (M+H)⁺; LCMS (Method B): T_(R=2.95) min.

Example 140

4-(((6S,6aS)-5-(((4-((S)—2-((S)—2-(((Allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicAcid (137)

As suspension of4-(5-((((4-((S)—2-((S)—2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)—2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoicacid (136) (1.2 g, 1.56 mmol) and Dess-Martin periodinane (1.35 g, 3.18mmol) in anhydrous dichloromethane (15 mL) was stirred at roomtemperature for 45 min. The reaction mixture was partitioned betweendichloromethane (2×50 mL) and saturated aqueous solution of sodiummetabisulfite (30 mL). The combined organic extracts were concentratedin vacuo. The resulting residue was purified by column chromatography(silica), eluting with methanol/dichloromethane (from 0% to 10%), togive the title compound (760 mg, 63%) as a yellow solid.

MS (ES+): m/z=767.8 (M+H)⁺; LCMS (Method B): T_(R=2.98) min.

Example 141

4-((S)—2-((S)—2-(((Allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(6S,6aS)-6-hydroxy-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(138)

A solution of4-(((6S,6aS)-5-(((4-((S)—2-((S)—2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoicacid (137) (440 mg, 0.573 mmol) in anhydrous dichloromethane (3 mL) wascharged withN—[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N—methylmethanaminiumhexafluorophosphate N—oxide (230 mg, 0.605 mmol) and anhydroustriethylamine (335 μL, 2.41 mmol). The reaction mixture was stirred atroom temperature for 30 min.4-Amino-1-methyl-N—(4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)-1H-pyrrole-2-carboxamidehydrochloride (128) (260, 0.578 mmol) was added and the resultingmixture was stirred at room temperature for 30 min. The reaction mixturewas quenched with a saturated aqueous solution of sodium hydrogencarbonate (20 mL) and extracted with dichloromethane (2×50 mL). Thecombined organic extracts were washed with water containing a few dropsof acetic acid (30 mL). The organic layer was then dried over sodiumsulfate, filtered and concentrated in vacuo. The resulting residue wasthen purified by column chromatography (silica), eluting withmethanol/chloroform (from 0% to 5%), to give the title compound (43 mg,6%) as a salmon solid.

MS (ES+): m/z=1163.1 (M+H)⁺; LCMS (Method B): T_(R=3.48) min.

Example 142

4-((S)—2-((S)—2-Amino-3-methylbutanamido)propanamido)benzyl(6S,6aS)-6-hydroxy-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(139)

To a solution of4-((S)—2-((S)—2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(6S,6aS)-6-hydroxy-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (138)(43 mg, 0.0370 mmol) in dichloromethane (1 mL) was sequentially addedtetrakis(triphenylphosphine)palladium(0) (2.1 mg, 5 mol %), andpyrrolidine (3.7 μL, 0.0450 mmol). The reaction mixture was stirred atroom temperature for 15 min. The reaction mixture concentrated in vacuoand subjected to high vacuum for 40 min until excess pyrrolidine wasremoved, to give the title compound (40 mg, 99%) as a yellow gum. Theproduct was carried through to the next step without any furtherpurification.

MS (ES+): m/z=1079.2 (M+H)⁺; LCMS (Method B): T_(R=2.90) min.

Example 143

4-((2S,5S)-37-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,11,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(6S,6aS)-6-hydroxy-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(140)

A solution of4-((S)—2-((S)—2-amino-3-methylbutanamido)propanamido)benzyl(6S,6aS)-6-hydroxy-2-methoxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate(139) (40 mg, 0.0370 mmol),1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oicacid (22.0 mg, 0.0370 mmol) andN—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (7.1 mg,0.0370 mmol) in anhydrous DCM (1 mL) was stirred at room temperature for30 min. The reaction mixture was directly purified by columnchromatography (silica), eluting with methanol/dichloromethane (from 0%to 15%), to give the title compound (41 mg, 67%) as a brown solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.95-9.67 (m, 3H), 8.15 (d, J=6.2 Hz, 1H),7.98 (d, J=5.1 Hz, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.77-7.64 (m, 3H),7.61-7.36 (m, 5H), 7.35-7.26 (m, 2H), 7.25-7.10 (m, 1H), 7.06-6.93 (m,3H), 6.68 (br. s., 1H), 5.76 (br. s., 1H), 5.10 (br. s., 1H), 4.86 (d,J=5.5 Hz, 1H), 4.44-4.30 (m, 1H), 4.25-4.15 (m, 1H), 4.11 (br. s., 1H),3.96-3.86 (m, 3H), 3.83-3.76 (m, 3H), 3.62-3.54 (m, 3H), 3.53-3.38 (m,28H), 3.30 (br. s., 15H), 3.26-3.20 (m, 1H), 3.18-3.08 (m, 2H), 2.86(br. s., 1H), 2.50 (br. s., 3H), 2.45-2.37 (m, 2H), 2.36-2.26 (m, 2H),2.10-1.86 (m, 3H), 1.62 (d, J=18.4 Hz, 1H), 1.53 (br. s., 2H), 1.29 (br.s., 3H), 1.25-1.05 (m, 3H), 0.94-0.76 (m, 6H); MS (ES+): m/z=1654.6(M+H)⁺; LCMS (Method B): T_(R=3.28) min; LCMS (Method A): T_(R=6.98)min.

Example 144

N—(4-((S)—2-((S)—2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)-4-(4-(4-(4-(((S)—2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(141)

A mixture of(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-valyl-L-alanine(160 mg, 0.420 mmol) and N—ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(150 mg, 0.600 mmol) in dichloromethane (10 mL) was stirred at 0° C. for30 min.(S)—N—(4-Aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(41) (300 mg, 0.400 mmol) in methanol (1 mL) was added to the reactionmixture at 0° C. The reaction mixture was stirred at 0° C. for 5 h andthen warmed to room temperature for another 11 h. The reaction mixturewas purified by neutral alumina column to afford the title compound (220mg, 50%) as a yellow solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.92 (s, 1H), 9.86 (s, 1H), 9.81 (s, 2H),8.15 (d, J=6.8 Hz, 1H), 8.00 (d, J=5.8 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H),7.68 (dd, J=22.8, 8.8 Hz, 4H), 7.56-7.42 (m, 5H), 7.38 (s, 1H), 7.27 (s,1H), 7.22 (s, 1H), 7.00-6.98 (m, 3H), 6.80 (s, 1H), 4.45-4.32 (m, 1H),4.22-4.09 (m, 2H), 4.07-3.97 (m, 2H), 3.90 (s, 3H), 3.82 (s, 6H),3.70-3.67 (m, 1H), 3.35-3.33 (m, 1H), 3.14-3.13 (m, 1H), 2.46-2.40 (m,2H), 2.16-2.14 (m, 2H), 2.09-2.01 (m, 3H), 1.96-1.90 (m, 1H), 1.89-1.81(m, 1H), 1.80-1.65 (m, 3H), 1.62-1.41 (m, 6H), 1.33-1.26 (m, 4H),1.26-1.12 (m, 6H), 0.92-0.76 (m, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ224.5, 217.8, 207.7, 204.1, 196.3, 195.7, 192.6, 192.0, 187.3, 185.2,183.3, 175.0, 172.7, 171.5, 171.4, 169.3, 166.8, 165.1, 160.0, 150.7,147.6, 143.0, 140.3, 137.6, 135.2, 134.9, 129.9, 126.6, 124.8, 122.5,120.8, 120.7, 119.8, 113.4, 111.8, 109.9, 106.7, 103.9, 75.4, 58.0,56.1, 54.1, 52.2, 49.4, 45.4, 37.4, 35.6, 32.3, 30.8, 28.2, 26.6, 25.3,23.0, 18.6, 18.5, 18.2, 15.3, 11.6; MS (ES+): m/z=1120.5 (M+H)⁺; MS(ES+): m/z=1120.5 (M+H)⁺; LCMS (Method B): T_(R=3.36) min.

Example 145

2,5-Dioxopyrrolidin-1-yl1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oate(143)

DCC (206 mg, 1.01 mmol) was added to a 0° C. solution of1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oicacid (142) (500 mg, 0.844 mmol), 1-hydroxypyrrolidine-2,5-dione (100 mg,0.886 mmol) in DCM/EtOAc (1/1, 20 mL). The reaction mixture was stirredat room temperature for 16 h. The reaction mixture was filtered andconcentrated to dryness under reduced pressure to give the titlecompound (500 mg, 85%) as a yellow oil. The product was carried throughto the next step without any further purification.

MS (ES+): m/z=689.3 (M+H)⁺; LCMS (Method B): T_(R=2.70) min.

Example 146

(9H-Fluoren-9-yl)methyl((S)—1-(((S)—1-((4-(4-(4-(4-(4-(((S)—2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(144)

A mixture of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl-L-alanine (325mg, 0.793 mmol) and EEDQ (326 mg, 1.32 mmol) in DMF (20 mL) was stirredat 0° C. for 1 h.(S)—N—(4-Aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(41) (500 mg, 0.661 mmol) was added to the reaction mixture and stirredat 0° C. for 5 h, then stirred at room temperature for 13 h. Thereaction mixture was diluted with DCM/TBME (1/8, 200 mL) and stirred for1 h before being filtered. The solid cake was dried under reducedpressure to give the title compound (500 mg, 66%) as a yellow solid. Theproduct was carried through to the next step without any furtherpurification.

MS (ES+): m/z=1149.2 (M+H)⁺; LCMS (Method B): T_(R=3.92) min.

Example 147

N—(4-((S)—2-((S)—2-Amino-3-methylbutanamido)propanamido)phenyl)-4-(4-(4-(4-(((S)—2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methy-1H-pyrrole-2-carboxamide(145)

A mixture of (9H-fluoren-9-yl)methyl((S)—1-(((S)—1-((4-(4-(4-(4-(4-(((S)—2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(144) (500 mg, 0.435 mmol) and piperidine (111 mg, 1.31 mmol) in DMF (10mL) was stirred at room temperature for 16 h. The reaction mixture wasdiluted with DCM/TBME (1/8, 200 mL) and stirred for 1 hour beforefiltered. The solid cake was dried under reduced pressure to give thecrude product (400 mg, 99% yield) as a yellow solid. The product wascarried through to the next step without any further purification.

MS (ES+): m/z=927.2 (M+H)⁺; LCMS (Method B): T_(R=2.81) min.

Example 148

N—(4-((2S,5S)-37-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)phenyl)-4-(4-(4-(4-(((S)—2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(146)

A mixture ofN—(4-((S)—2-((S)—2-amino-3-methylbutanamido)propanamido)phenyl)-4-(4-(4-(4-(((S)—2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(145) (400 mg, 0.431 mmol), 2,5-dioxopyrrolidin-1-yl1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oate(143) (357 mg, 0.518 mmol) and DIPEA (143 μL, 0.865 mmol) in DMF (10 mL)was stirred at room temperature for 16 h. The reaction mixture wasconcentrated under reduced pressure. The resulting residue was thenpurified by column chromatography (silica), eluting withmethanol/dichloromethane (from 0% to 10%), followed by preparative TLC,to give the title compound (100 mg, 15%) as a yellow gum.

¹H NMR (400 MHz, DMSO-d₆) δ 9.95 (s, 1H), 9.88 (s, 1H), 9.81 (d, J=3.5Hz, 2H), 8.17 (d, J=7.2 Hz, 1H), 8.05-7.97 (m, 1H), 7.90 (d, J=8.6 Hz,1H), 7.71 (d, J=8.4 Hz, 2H), 7.66 (d, J=8.9 Hz, 2H), 7.55 (d, J=8.9 Hz,2H), 7.49 (d, J=8.4 Hz, 2H), 7.44 (s, 1H), 7.40 (s, 1H), 7.22 (s, 1H),7.02-6.93 (m, 2H), 4.63-4.47 (m, 1H), 4.46-4.31 (m, 1H), 4.27-4.15 (m,1H), 4.11 (q, J=5.2 Hz, 3H), 4.06-3.97 (m, 1H), 3.90 (s, 3H), 3.86-3.80(m, 3H), 3.74-3.64 (m, 3H), 3.64-3.55 (m, 4H), 3.52-3.46 (m, 22H),3.19-3.10 (m, 8H), 2.67 (s, 1H), 2.46-2.38 (m, 3H), 2.32 (t, J=7.2 Hz,2H), 2.11-1.92 (m, 4H), 1.92-1.69 (m, 2H), 1.69-1.48 (m, 3H), 1.31 (d,J=7.1 Hz, 3H), 1.23 (br. s., 6H), 0.86 (dd, J=6.7, 15.5 Hz, 6H); MS(ES+): m/z=1501.9 (M+H)⁺; LCMS (Method B): T_(R=3.13) min.

Example 149

tert-Butyl(5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate(147)

A solution of4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylicacid (126) (300.00 mg, 0.68 mmol) in N,N—dimethylformamide (8 mL) wascharged with N,N—dimethylpyridin-4-amine (250.7 mg, 2.0 mmol) andN—(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (327.8 mg,1.71 mmol) and it was stirred for 30 min at room temperature. To thereaction mixture, benzene-1,4-diamine (88.6 mg, 0.82 mmol) was thenadded and the solution was stirred for further 18 h. The reactionmixture was quenched with a saturated aqueous solution of sodiumhydrogen carbonate (30 mL) and loaded with brine (80 mL). The aqueousphase was extracted with ethyl acetate (2×60 mL). The combined organicextracts were concentrated in vacuo. The resulting residue was purifiedby column chromatography (silica), eluting with acetone/dichloromethane(from 0% to 40%), to give the title compound (355 mg, 99%) as a brownoil.

MS (ES+): m/z=529 (M+H)⁺; LCMS (Method B): T_(R=3.28) min.

Example 150

4-Amino-N—(4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)-1-methyl-1H-pyrrole-2-carboxamide(148)

To a solution of tert-butyl(5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate(147) (100.0 mg, 0.19 mmol) in 1,4-dioxane and methanol (1:1) (2 mL)hydrochloric acid (4 M in 1,4-dioxane) (2 mL) was added drop wise. Thereaction mixture was stirred for 4 h and then quenched through theaddition of a 1 M solution of sodium hydroxide aqueous solution (10 mL,10 mmol). The mixture was then diluted with brine (30 mL) and theresulting aqueous phase was washed with dichloromethane (3×30 mL). Theorganic layer was dried over magnesium sulphate anhydrous, filtered andconcentrated in vacuo affording the titled compound (70 mg, 86%) as abrown oil.

¹H NMR (400 MHz, CD₃OD) δ 7.58-7.54 (m, 3H), 7.53-7.49 (m, 2H),7.35-7.30 (m, 2H), 7.22 (s, 2H), 6.75-6.71 (m, 2H), 6.61 (s, 1H), 3.91(s, 3H), 3.83 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 126.5, 124.9, 124.6,123.1, 122.7, 121.0, 115.3, 110.0, 35.6, 35.2; MS (ES+): m/z=429 (M+H)⁺;LCMS (Method B): T_(R=2.35) min.

Example 151

Evidence of DNA Adduct Formation by HPLC

Interaction of C8-linked PDD monomers with duplex transcription factorconsensus sequence was studied with an HPLC assay utilizing a X-bridgeMS C18 2.5 μM OST column (2.3×50 mm) and a gradient of 40%acetonitrile/water and 100 mM TEAB (Tetraethylammonium bromide)/water asmobile phase with a flow rate of 0.5 mL/min and UV detection at 254 nm.A 4:1 molar ratio of ligand:oligonucleotide was used, with eachsingle-stranded oligonucleotide dissolved in 1 M ammonium acetate toform stock solutions of 1 mM. The oligonucleotides were initiallyannealed by heating their 1 mM solutions to 70° C. for 10 mins followedby gradual cooling over 8 hours and storage overnight at −20° C. Workingsolutions of oligonucleotides of 25 μM were then prepared by dilutingthe annealed stock solutions with 100 mM ammonium acetate. The ligandswere dissolved in DMSO to form a stock solution of 10 mM which wasstored at −20° C. for no longer than four months. Working solutions ofthe drug of 100 μM were prepared by diluting the stock solution with 100mM ammonium acetate. The working solutions of the ligands were added tothe working solution the oligonucleotides at RT, and the mixtureincubated for different time intervals at room temperature.

Example 152

Fluorescence Resonance Energy Transfer (FRET) Assay

Oligonucleotide sequences used for the FRET assays were purchased fromEurogentec, Southampton, UK: TAMRA (6-carboxytetramethylrhodamine) andFAM (6-carboxyfluorescein) are acceptor and donor fluorophores,respectively. From 20 μM stock solutions, 400 M solutions in FRET buffer(optimized as 50 mM potassium, 50 mM cacodylate, pH 7.4) were preparedprior to use. The oligonucleotides were annealed through heating thesamples to 90° C. for 10 mins followed by cooling to room temperatureand storing at this temperature for 5 h. Dilutions from the initial 5 mMDMSO stock solution were performed using FRET buffer. Annealed DNA (50μL) and sample solution (50 μL) were added to each well of a 96-wellplate (MJ Research, Waltham, Mass.), and processed in a DNA EngineOpticon (MJ Research). Fluorescence readings were taken at intervals of0.5° C. over the range 30-100° C., with a constant temperaturemaintained for 30 seconds prior to each reading. Incident radiation of450-495 nm was used, with detection at 515-545 nm. The raw data wereimported into the program Origin (Version 7.0, OringinLab Corp.), andthe graphs were smoothed using a 10-point running average, and thennormalized. Determination of melting temperatures was based on values atthe maxima of the first derivative of the smoothed melting curves usinga script. The difference between the melting temperature of each sampleand that of the blank (ΔTm) was used for comparative purposes.

TABLE 1 ΔTm determined after 24 hours incubation with TranscriptionFactor duplex DNA sequences ΔTm at 1 μM ligand concentration AP−l AP−lNFκB NFκB (2^(nd) (1^(st) (2^(ND) Compound (1^(st) transition)transition) transition) transition) 13 12 23 11 19 17 11 26 13 18 20 912 8 13 24 10 14 9 15

Example 153

Cytotoxicity Analysis of C8-Linked PDD Monomers by MTT Assay

Cell Culture

MDA MB231 (triple negative human breast cancer) was obtained from theAmerican Type Culture Collection. The cell-line was maintained inmonolayer culture in 75 cm² flasks (TPP, Switzerland) under a humidified5% CO₂ atmosphere at 37° C. The MDA MB231 cell line was maintained inhigh glucose DMEM (4.5 g\l; Invitrogen), foetal bovine serum (10%,Biosera UK), non-essential amino acids (1×; Invitrogen), L-glutamine (2mM; Invitrogen) and Penicillin-Streptomycin (1% v/v, Invitrogen). TheHeLa cell line was maintained in Dulbecco's Modified Eagles Media (DMEM;Invitrogen) supplemented with foetal bovine serum (10% v/v; Invitrogen),L-glutamine (2 mM; Invitrogen), non-essential amino acids (ix;Invitrogen) and Penicillin-Streptomycin (1% v/v, Invitrogen). Forpassaging, cells were washed with PBS (GIBCO 14040, Invitrogen, UK),incubated with trypsine (GIBCO 25300, Invitrogen, UK), and re-seededinto fresh medium. For seeding, cells were counted using a Neubauerhaemocytometer (Assistant, Germany) by microscopy (Nikon, USA) on anon-adherent suspension of cells that were washed in PBS, trypsinised,centrifuged at 8° C. at 8000 rpm for 5 min and re-suspended in freshmedium.

MTT Assay

The cells were grown in normal cell culture conditions at 37° C. under a5% CO₂ humidified atmosphere using appropriate medium. The cell countwas adjusted to 10⁵ cells/ml and 5,000-20,000 cells were added per welldepending on the cell line. The cells were incubated for 24 hours and 1μl of the appropriate inhibitor concentrations were added to the wellsin triplicates. After 72 h of continuous exposure to each compound, thecytotoxicity was determined using the3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)(Lancaster Synthesis Ltd, UK) colorimetric assay.^([34) Absorbance wasquantified by spectrophotometry at λ=570 nm (Envision Plate Reader,PerkinElmer, USA). IC₅₀ values were calculated by a dose-responseanalysis using the GraphPad Prism® software.

TABLE 2 IC₅₀ values (nM) determined after 72 hours exposure for theC8-linked PDD monomers. IC₅₀ (nanomolar) MDA MB 231 (Triple negativeHeLa breast cancer cell (Cervical cancer cell Compound line) line) 13 64± 9.6  0.6 ± 0.4 17 21 ± 1.8  1.2 ± 0.8 20 0.3 ± 0.22 0.14 ± 0.09 24 0.8± 0.66   1 ± 0.12

Example 154

Biological and Biophysical Characterisation of Free Payloads In VitroCytotoxicity

The in vitro cytotoxicity of compounds 73, 76, 81, 88 and 93 wereevaluated in the JIMT-1, MCF-7, MDA-MB-453 and SK-BR-3 (all breastcancer) cell lines using the standard MTT assay for a 72 hour incubationperiod (Table 3).

TABLE 3 Cytotoxicity of 73, 76, 81, 88 and 93. Cytotoxicity (nM)Compound MDA-MB- Number JIMT-1 MCF-7 453 SK-BR-3 73 0.33 0.42 0.20 0.2788 0.37 0.60 0.13 0.06 76 0.43 0.53 0.19 0.29 81 0.50 1.40 0.42 0.33 930.37 2.00 0.18 0.09

Compound 41 was evaluated in a broader cell-line panel (Table 4)affording low picomolar activity in a variety of cancer types,suggesting broad use for the payload class.

TABLE 4 In vitro cytotoxicity of 41 in a broad panel of cancercell-lines (72 hour incubation). IC₅₀ (Nanomolar, 72 hour Cell Lineincubation) 786-O (Renal Cell, CD70) 0.53 SK-HEP-1 (Liver) 0.32 SK-MEL-5(Melanoma) 0.39 Calu-3 (Lung) 0.65 A549 (Lung) 0.18 AGS (Gastric) 0.07PC₃ (Prostate) 0.36 SW480 (Colorectal) 0.29 EC Cancer Stem Cell (Cancerstem cells) 2.64 AML2 (AML) 0.016 HL60 (APML) 0.009 LnCap (Prostate)0.47 BxPC3 (Pancreas) 0.43 A375 (Skin) 0.28

Example 155

DNA Cross-Linking Assay

The ability of 41 to cross-link DNA was evaluated using an assayinvolving a linear double-stranded TyrT fragment (FIG. 5). The PBD dimerTalirine (SGD1882) was used as a positive control, as PBD dimers havepreviously been shown to cross-link DNA⁽³²⁾. Following denaturationconditions (treatment with formamide and heating at 65° C. for 5 min),the DNA strands were completely separated (see control C2, FIGS. 6 and7). The presence of an interstrand cross-link holds the denaturedstrands in close proximity, and cross-linked adducts therefore run asdouble-stranded DNA on polyacrylamide gel.

Both compounds were tested at six different concentrations, and theassay was repeated twice. The cross-linking ability of 41 is shown inFIG. 6. Cross-links are not detectable at any concentration (i.e., from10 μM to as low as 0.1 nM), whereas the PBD dimer Talirine producescross-links at concentrations as low as 10 nM (FIG. 7). These resultsdemonstrate that 41 is incapable of cross-linking DNA, consistent withits proposed mono-alkylation mechanism of action.

Example 156

DNA Footprinting

The DNA sequence selectivity profile of selected molecules wasinvestigated using a modification of the previously established DNAfootprinting assay^([32]). Following an overnight incubation of theligand-DNA complexes, the mixture was mixed with strand separationbuffer containing 10 mM EDTA, 10 mM NaOH, 0.1% bromophenol blue, 80%formamide and incubated at 100° C. for 3 min. The mixture was thenimmediately cooled on ice and run on an 8% denaturing gel. Examinationof the obtained gel (FIGS. 8A-C) shows footprints produced by themolecules on MS1 and HexA DNA sequences. FIGS. 8A and 8B shows DNAfootprint illustrating the interaction of 73 (B), 76 (D), 81 (E), 88 (C)and 93 (G) with the MS1 DNA fragment and HexA DNA fragment, respectively(lanes A and F represent further controls). FIG. 8C shows a DNAfootprint illustrating the interaction of the PBD dimer Talirine withthe MS1 DNA fragment. Interestingly, although the MS1 and HexA DNAfragments contain multiple potential binding sites for molecules 73, 76,81 and 88 (i.e., multiple examples of potential G-alkylating sites),only six preferred sites in the case of MS1 and five in the case of HexAwere observed during this experiment. 93 was found to bind to twofurther sequences in MS1 not occupied by 73, 76, 81 and 88, suggesting adegree of sequence interactivity not present in other molecules in theclass. Molecular modelling studies suggest that this occurs due to thering nitrogen of the imidazole group of 93 forming sequence-interactiveH-bonds with guanine bases (data not presented). These data also suggestthat the molecules all act in a highly sequence selective manner with adifferent sequence selectivity profile to the PBD dimer Talirine (greenblocks, FIG. 9). The possible adducts formed within the MS1 and HexAsequences are shown in FIGS. 9 and 10 respectively. FIG. 9 showscompounds 73, 41, 76, 81, 88 (all represented in cyan) and 93(represented in black) all bind to a similar binding site. 93 (solidblack line) also interacts with two further sites. The PBD dimerTalirine (green) illustrates a different binding pattern to 73, 76, 81,88 and 93. Strong DNA footprints are represented by solid lines andweaker footprints are represented by hatched lines. FIG. 10 showscompounds 73, 41, 76, 81 and 88 (all represented in cyan) and 93(represented in black) all bind to similar binding sites. Strong DNAfootprints are represented by solid lines, and weaker footprints arerepresented by hatched lines

Example 157

FRET DNA Melting

FRET DNA melting studies were undertaken on a fluorescently labelledduplex DNA sequence reacted with 41 and fragmented forms of the molecule(i.e, using intermediates 106, 107 and 148). The sequence (Figure to)was designed to provide additional evidence that 41 can effectivelystabilise a particular DNA sequence derived through the Footprintingstudies (i.e., XGXWWWW where X represents any base, G represents guanineand W indicates adenine or thymine). FIG. 11 shows fluorescentlylabelled DNA duplex used in the FRET melting study to study thestabilisation of DNA by 41, 106, 107 and 148. The labels werefluorescein (F) and dabcyl (Q).

FIGS. 12A and B show FRET Denaturation data for 5′-AAAAAAAGAAATTTAAA-3′when bound to 41 (FIG. 12A), 106, 148 and 107 (FIG. 12B, right to left).The melting temperature of the duplex increases significantly inproportion to the concentration of 41 present, providing strongsupporting evidence that this compound can effectively stabilise DNA.This is in contrast to data derived for the fragments (lower panel),where little stabilisation is observed. The mono-alkylated adduct formedby 41 stabilizes the duplex form, producing very large increases inmelting temperature (i.e. T_(m) values) of >35° C. for5′-AAAAAAAGAAAAAATTT-3′ (FIG. 12A) indicating a very high bindingaffinity for this particular sequence. Interestingly, when fragmentedinto its component parts (i.e., 106, 107 and 148), the fragments exhibitlittle DNA stabilisation (FIG. 12B), suggesting that the enhanced DNAstabilisation and potent cytotoxicity occurs due to the uniquecombination of the fragments.

Example 158

Transcription Factor Plate Array Assay

A transcription factor plate array assay experiment was undertaken toestablish which transcription factors are down-regulated through thealkylation of DNA by 41. The study showed that the major transcriptionfactors down-regulated were NFAT, NF-κB, OCT-4 and GATA. FIG. 13A showsa graph illustrating percentage difference in TF activation in treatedcells and FIG. 13B shows the percentage difference in TF activation incells not treated with 41. FIG. 14 shows a graph illustrating a summaryof the major transcription factors up- and down-regulated by 41. Theconsensus sequences of each of these transcription factors correspondsto the DNA footprinting pattern observed for 41.

41 has been found to bind to XGXWWWW where X is any base, G representsguanine and W is A or T. In the case of the transcription factor GATA(consensus site WGATAR, where R is A or G), an obvious binding sitematching to the footprint is evident. Similarly, in the case of NF-κB(consensus site GGGRRNNYYCC where N is any base, and Y is C or T), abinding site (bold and underlined) can be identified, and the consensussequence of NFAT (GGGAA) also directly corresponds to the DNA footprint.

Summary of Examples 154 to 158

Taken together, the biophysical data presented above provide strongevidence that 73, 41, 76, 81, 88 and 93 effectively stabilise DNA with ahigh degree of sequence-specificity. Furthermore, when fragmented intoits component parts, the individual fragments of 41 exhibit a low degreeof DNA stabilisation when reacted with DNA, but provide a large degreeof stabilisation when joined, highlighting the uniqueness of the parentstructure to recognize specific DNA sequences. Together, these datasuggest that the population of DNA adduct types derived may account forthe cytotoxicity of this family of compounds in cells. Furthermore, DNAFootprinting studies indicate a degree of sequence selectivity for theclass, with the DNA-binding site generally corresponding to XGXWWWWwhere X represents any base and W indicates adenine or thymine. 41 wasshown to down-regulate a number of key transcription factors (e.g.,NF-κB and GATA), and analysis suggests that their binding sitescorrespond to the main DNA Footprint observed for this class ofmolecules. Overall, these data suggest that the potent cytotoxicityobserved for the PDD class of payloads is directly related to theirDNA-binding affinity and sequence selectivity which can result in theinhibition and down-regulation of key transcription factors. The factthat these compounds mono-alkylate rather than cross-link DNA as occurswith the PBD dimers, suggests that they may produce less overallsystemic toxicity³, and may provide a higher Therapeutic Index in animalmodels or human clinical trials.

Example 159

γH2aX Assay

The γH2aX assay was used to evaluate the level of DNA damage caused by41 and Talirine. FIG. 13 shows a graph illustrating percentagedifference in TF activation in treated cells versus cells not treatedwith 41. FIG. 14 shows a graph illustrating a summary of the majortranscription factors up- and down-regulated by 41. FIGS. 15A and B showgraphs illustrating cell cycle arrest by 41 (FIG. 15A) and Talirine(FIG. 15B), where MEC represents Minimum Effective Concentration thatsignificantly crosses the vehicle control threshold, AC50 represents theconcentration at which 50% maximum effect is observed for each cellhealth parameter, and MR is the maximum response.

The results show an equal amount of DNA damage (FIG. 13) at similarconcentrations, despite the fact the PBD dimer cross-links DNA, but 41,and analogues, only mono-alkylate DNA. This degree of DNA damage mayprovide a rationale for the potent cytotoxicity observed for 73, 41, 76,81, 88 and 93.

Example 160

Cell Cycle

FIG. 16 shows a graph outlining cell cycle arrest induced by 41. Studieson the arrest of cell cycle indicate that 41 arrests at the G0/G1-Sphase, a distinctly different mechanism of action to otherDNA-interactive agents (e.g., PBD dimers^([33]) and the IGNmono-alkylator^([34]) which arrest cell cycle at the G2-M phase).

Examples 161

Conjugation of Compound 141 to IgG1 Antibody (Forming ADC1)

141 was conjugated to an IgG1 antibody targeted to Antigen X in astochastic manner. FIG. 17 shows an SEC profile of Antibody X. 98.9%monomer, 1.0% dimer, and 0.1% LMW as indicated. The peak at about 23minutes originates from the formulation of the antibody. FIG. 18 showsan HIC profile of Antibody X. FIG. 19 shows a PLRP trace of Antibody X.Heavy (H0) and light (L0) chain peaks as indicated.

The antibody was of good quality with 98.9% monomer content (FIG. 17)and a single peak with a small shoulder on HIC (FIG. 18). PLRP showedthe expected pattern for reduced Light and Heavy chain. The minor peakseluting after the main L0 and H0 are likely the result of intrachaindisulphide reduction (FIG. 19).

DAR (Drug Antibody Ratio) assignment was possible through PLRP analysis(FIG. 21; average DAR calculated as 1.8 with the light/heavy chainspecies assigned as indicated) and is in good agreement with the valuecalculated by HIC using the integration highlighted in FIG. 20 (averageDAR calculated as 1.9 with the DAR species assignments as indicated).The conjugation process caused no significant aggregation compared tothe starting antibody. Dimer level was increased by 2.4%, but LMWremained the same. FIG. 22 shows an SEC profile of IgG1-141; 96.5%monomer, 3.4% dimer, 0.1% HMW as indicated No free toxin linker could bedetected in the ADC sample (see FIG. 23).

Example 162

Conjugation of 82 to Trastuzumab (Forming ADC2)

141 was successfully conjugated to Trastuzumab (stochastic conjugation,DAR 1.9) and an isotype control antibody. DAR assignment was possiblethrough HIC (FIG. 24A) and SEC analysis (FIG. 24B). Littlehydrophobicity was observed.

Example 163

Site-Specific Conjugation (Forming ADC3)

141 was conjugated to a THIOMAB®-based version of Trastuzumab (DAR 2).The THIOMAB antibody had a relatively low monomer content of 92.4% with6.7% dimer (see FIG. 25). This was not unexpected and has been seenbefore with this antibody due to the formation of inter moleculardisulfide bonds with the mutated cysteines. The HIC and PLRP profilesfor the THIOMAB are shown in FIGS. 26 and 27 respectively.

The THIOMAB-141 conjugate resolves reasonably well by hydrophobicinteraction chromatography. An identification of DAR species is possibleby using the relative absorbance at 280 and 330 nm to identify specieswith different drug loading or isomers of the same drug loading. The DAR2 species appears in two overlapping peaks (same DAR confirmed byspectral comparison), and are almost fully resolved from the DAR 0.There is probably some DAR₁ species as there is an inflection at theposition this is expected; DAR 1 species clearly observed duringdevelopment when underconjugation was achieved. The PLRP profile shows ahigh degree of site specific conjugation and a small amount ofnon-specific conjugation to the heavy chain because of incompletere-oxidation of all S—S bonds; typical of all THOMAB processes. Themonomer level is higher than the starting antibody due to reduction ofinter molecular S—S bonds. FIG. 28 shows the HIC profile of THIOMAB-141.Average DAR calculated as 1.9 with the DAR species assignments asindicated. FIG. 29 shows the PLRP trace of THIOMAB-141. Average DARcalculated as 1.8 with the light/heavy chain species assigned asindicated. The peak labelled as “H1?” might contain L2 species. Theconjugation process caused a sizable amount of dimerization and theformation of a small amount of high molecular weight aggregates (HMW).FIG. 30 shows the SEC profile of THIOMAB-141; 94.8% monomer, 4.8% dimer,0.4% HMW as indicated.

Example 164

In Vitro Cytotoxicity of ADCs

The resulting ADCs were evaluated in vitro against relevant antigenpositive cell-lines. In the case of the ADC targeted to Antigen X(ADC1), potent cytotoxicity was observed.

TABLE 5 Summary of in vitro cytotoxicity data derived for IgG1- basedADC targeted to Antigen X using 41 as the payload. IC50 (nM) AntigenAntigen Positive Cell- Positive Cell- Line 1 Line 2 IgG1-based ADC 5 day0.67 0.47 (Average DAR = 2) incubation

In the case of the trastuzumab-based ADC (ADC2), the ADC possessedpotent activity in the antigen-positive cell-line (i.e., SK-BR-3) and nocell-killing was observed in both the JIMT-1 and MCF-7 cell-linesindicating the targeted effect of the ADC. Furthermore, potency of thefree payload was enhanced through attachment to the antibody.

TABLE 6 Summary of in vitro cytotoxicity data derived fortrastuzumab-based ADC using 41 as the payload. IC50 (nM) (72 hourincubation ZR75-1 (HER2++) SK-BR-3 (Trastuzumab MCF-7 (HER2+++)resistant) (HER2−) Trastuzumab THIOMAB ®- 0.009 28.8 82.3 based ADC(average DAR = 2) IgG1 control ADC 18 122.2 157 (average BAR = 2) FreePayload 0.086 0.63 0.289

Finally, the THIOMAB®-based ADC (ADC3) possessed potent activity in theantigen-positive cell-line (i.e., SK-BR-3) and limited cell-killing wasobserved in both the ZR₇₅-1 and MCF-7 cell-lines indicating the targetedeffect of the ADC. Furthermore, potency of the free payload was enhancedthrough attachment to the antibody.

Example 165

In Vivo Tolerability of ADCs

The maximum tolerated dose for ADC 3 was established as 9 mg/kg. The PBDdimer-based THIOMAB® ADC was found to have an MTD of ˜4 mg/kg,suggesting enhanced tolerability of the mono-alkylating payload. TheTHIOMAB®-141-based ADC possesses similar tolerability to thenon-alkylating MMAE-based ADC. In this instance, loss of 15% of bodyweight was considered a toxic dose. FIG. 31 shows a graph illustratingdose tolerability of 141, PBD dimer (Talirine) and MMAE-based ADCs. TheMTDs of the ADCs conjugated in a stochastic manner (i.e., ADCs 1 and 2)were found to be ˜6 mg/kg.

Example 166

In Vivo Efficacy

FIG. 32 shows a graph illustrating mean tumour volume versus time aftertwo doses of ADC 2 (Day 0 and Day 7). ADC2 was found to be highlyefficacious in a cancer-derived xenograft model (in mice) expressingTarget X, where complete tumour remission was observed out to a periodof 38 days after two doses of the ADC. FIG. 33 shows a graphillustrating mean tumour volume of a PDX model versus time after twodoses of ADC 2 (Day 0 and Day 14). Similarly, in the case of a PDX modelexpressing the same target, cytostasis was observed at 0.5 mg/kg aftertwo doses.

Example 167

Materials and Methods for Examples 154 to 166

DNA Fragments and Footprinting

The preparation of the TyrT DNA fragment (FIG. 5) has been previouslydescribed^([35]). Briefly, the sequence which had been cloned into theBamHI site of pUC18 was obtained by cutting with HindIII and EcoRI.Radiolabelled DNA fragments were prepared by filling in the 3′-end ofthe HindIII site with [α-³²P]dATP using Klenow DNA polymerase (exo−).

The radiolabelled DNA fragment was separated from the remainder of theplasmid DNA on a 6% non-denaturing polyacrylamide gel. The gel (20 cmlong, 0.3 mm thick) was run at 400 V in 1×TBE running buffer for about1-2 h, until the bromophenol blue had run most of the way down the gel.The glass plates were separated and the position of the labelled DNAfragment was established by short (1 min) exposure to an X-ray film. Therelevant band was then cut from the gel and the radiolabelled DNA elutedby adding 300 μL 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA andgently agitating overnight at room temperature. The eluted DNA wasfinally precipitated with ethanol and re-suspended in a suitable volumeof 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA buffer so as to give atleast to counts per second/μL on a hand-held Geiger counter. With freshplasmid and α-³²P-dATP this process typically generated about 150 μL ofradiolabelled fragment DNA. The absolute concentration of the DNA is notimportant, and it is typically lower than to nM.

Footprinting reactions were performed as previously described^([36])using the DNA fragments HexA and HexB, which together contain all 64symmetrical hexanucleotide sequences^([6]), and MS1 that contains allpossible 134 tetranucleotide sequences^([38]). The DNA fragments wereobtained by cutting the parent plasmids with HindIII and SacI (for HexAand MS1) or EcoRI and PstI (for HexB), and were labelled at the 3′-endof the HindIII or EcoRI sites with [α-³²P]dATP using reversetranscriptase or exo− Klenow fragment. After gel purification, theradiolabelled DNA was dissolved in 10 mM Tris-HCl pH 7.5 containing 0.1mM EDTA, at a concentration of about to c.p.s per μL as determined on ahand held Geiger counter. 1.5 μL of radiolabelled DNA was mixed with 1.5μL ligand that had been freshly diluted in 10 mM Tris-HCl pH 7.5,containing 10 mM NaCl. The complexes were left to equilibrate for atleast 12 hours before digesting with 2 μL DNase I (final concentrationabout 0.01 units/mL). The reactions were stopped after 1 minute byadding 4 μL of formamide containing 10 mM EDTA and bromophenol blue(0.1% w/v). The samples were then heated at 100° C. for 3 minutes beforeloading onto 8% denaturing polyacrylamide gels containing 8 M urea. Gelswere fixed in 10% acetic acid, transferred to 3MM paper, dried andexposed to a phosphor screen overnight, before analysing with a Typhoonphosphorimager

Compounds

73, 41, 76, 81, 88 and 93 were synthesised as described above and thePBD dimer Talirine was obtained from a commercial source. Stock solutionwas prepared by dissolving the ligands in DMSO to give a concentrationof 10 mM. From this stock solution, working solutions of the desiredconcentration were prepared by diluting with 10 mM Tris-HCl, pH 7.5containing 10 mM NaCl.

Cross-Linking Assay

Radiolabelled DNA (1.5 μL) was mixed with 1.5 μL ligand solution ofvarious concentrations (10 μM-10 nM) and incubated overnight at 37° C.

After overnight incubation, the samples were mixed with 7 μL loadingsolution (80% formamide containing 10 mM EDTA, 10 mM NaOH, 0.1%bromophenol blue) and incubated at 65° C. for 5 min. Control 1 (C1) fornative double-stranded DNA consisted of 1.5 μL labelled DNA, 1.5 μL 10mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and 7 μL ix loading dye.Control 2 (C2) for denatured native single-stranded DNA was composed of1.5 μL labelled DNA, 1.5 μL 10 mM Tris-HCl, pH 7.5 containing 0.1 mMEDTA which was incubated at 65° C. for 5 min. Control 3 (C3) for nativedouble-stranded DNA consisted of 1.5 μL labelled DNA, 1.5 μL 10 mMTris-HCl, pH 7.5 containing 0.1 mM EDTA and 7 μL SSB. Control 4 (C4) fordenatured native single-stranded DNA was composed of 1.5 μL labelledDNA, 1.5 μL 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and 7 μL SSBwhich was incubated at 65° C. for 5 min. Separation was performed on a7.5% denaturing polyacrylamide gel (20 cm long, 0.3 mm thick) at 500Vfor about 4 h until the dye reached the bottom of the gel. The gelplates were then separated, the gels fixed by immersing in 10% (v/v)acetic acid, followed by transfer to Whatmann 3MM paper and drying undervacuum at 80° C. The dried gel was then exposed to a phosphorimagerscreen overnight before scanning using a Typhon FLA 7000 instrument.

FRET Studies Methodology

Oligonucleotides were obtained from ATDbio (Southampton, UK) inlyophilised form. They were labelled with a fluorophore molecule(F=fluorescein) at the 5′-end and a quencher molecule (Q=dabcyl) at the3′-end of the complementary strand. Each oligonucleotide was dissolvedin distilled H₂O to form stock solutions of 100 μM. Working solutions of5 μM were prepared by diluting the stock solution with distilled H₂O.

The following buffers were used: 250 mM phosphate buffer pH 7.4(consisting of sodium dihydrogen phosphate and sodium phosphate dilutedin distilled H₂O) and 5 M sodium chloride buffer. All buffers anddistilled H₂O were filtered through a 0.2 μM filter prior to use.

For the FRET experiments stock solutions of 41, 106, 107 and 148 wereprepared by dissolving the compounds in DMSO to give a concentration of10 mM. From this stock solution, working solutions of the desiredconcentration were prepared by diluting the stock solution withdistilled H₂O.

The reaction mixture was comprised of 4 μL of 250 mM phosphate buffer(final concentration of 50 mM), 4 μL flourophor and 4 μL quenchermolecule of the appropriate oligonucleotide for a final concentration of0.2 μM, 4 μL 5 M sodium chloride (final concentration of 1 M NaCl), and4 μL of distilled H₂O. This mixture was heated in an Eppendorf tube at90° C. for 1 min and slowly cooled down to room temperature. Thisprocess was carried out to anneal the single strands to double-strandedDNA. Following this, 4 μL of the ligand was added in the desiredconcentration and the mixture incubated overnight either at roomtemperature or 4° C. A control sample of DNA only was prepared by mixing4 μL 250 mM phosphate buffer (final concentration of 50 mM) with 4 μLfluorophore-labelled and 4 μL quencher-labelled oligonucleotides (of theappropriate sequence) to give a final concentration of 0.2 μM, 4 μL 5 Msodium chloride (final concentration of 1 M NaCl) and 4 μL distilledH₂O. This mixture was analysed without prior annealing.

Fluorescence melting profiles were measured using a Roche LightCyclerusing a total reaction volume of 20 μL. Initially, the samples weredenatured by heating to 95° C. at a rate of 1° C. min⁻¹. The sampleswere then maintained at 95° C. for 5 min before annealing by cooling to25° C. at 1° C. min⁻¹. The samples were then held at 25° C. for afurther 5 min and finally melted by heating to 95° C. at 1° C. min⁻¹.Annealing steps and melting steps were all recorded and changes influorescence were measured at 520 nm.

T_(m) values were obtained from the first derivates of the meltingprofiles using the Roche LightCycler software.

MTT Cytotoxicity (Example 154)

Tumor cell lines were maintained in RPMI1640 medium supplemented with10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and 1 mMsodium pyruvate. 1800 cells per well were seeded in a volume of 180 μlin a 96-well flat bottom polystyrene plate. The cells were allowed toadhere overnight at 37° C. in a CO₂ incubator. Ligands were initiallyformulated in DMSO, and stocks stored at −80° C. They were then furtherformulated at 10× concentration in RPMI1640 medium. 20 ul of dilutedsamples were added into each treatment well. On each plate, blank wellswith no cells, and untreated wells containing cells, were included.Plates were then cultured at 37° C. in a CO₂ incubator for 72 hrs.Cytotoxicity was evaluated using a tetrazolium salt-based assay, the MTTassay. After 72 hours, the supernatant was removed from each well and200 μl of a sterile filtered 500 μg/ml MTT solution in water added toeach well. The plates were then incubated at 37° C. in a CO₂ incubatorfor 4 hrs. The supernatant was then removed and the formazan crystalsformed solubilized by adding 150 μl of DMSO to each well. The plate wasthen read on a plate reader at 540 nm, and percentage cell survivalcalculated as follows: ((mean absorbance treated wells at concentrationx−mean absorbance blank wells)÷(mean absorbance untreated wells atconcentration x−mean absorbance blank wells))×100. Data were plotted asconcentration in nM vs. % cell survival in Microsoft Excel, and IC₅₀values (concentration where cell survival is reduced by a half) weredetermined from the graph.

γH2ax Assay and Cell Cycle Arrest

HepG2 cells were plated on 96-well tissue culture treated black walledclear bottomed polystyrene plates, 100 μL per well. The cells were dosedwith test compound at a range of concentrations. At the end of theincubation period, the cells were loaded with the relevant dye/antibodyfor each cell health marker. The plates were then scanned using anautomated fluorescent cellular imager, ArrayScan® (Thermo ScientificCellomics).

Cytotoxicity and DNA damage were assessed using a multi-parametricapproach using High Content Screening (HCS). Decreased cell count is adirect indication of toxicity, but many molecules cause sub-lethaltoxicities that do not cause changes in cell viability over theincubation period. This assay scores compounds across direct andindirect measures of toxicity. An increase in DNA damage (p-H2AX)indicates a rise in the number of double strand breaks (DSBs). DSBscause the phosphorylation of the histone H2AX at Ser139. DSBs are anindication of genotoxicity and can lead to apoptosis (programmed celldeath). Cell cycle arrest was determined as the ratio of G0/G1(2N) toG2/M(4N), where an increase is linked to G0/G1 arrest and a decreases islinked to G2/M arrest.

Transcription Factor Plate Array Assay

The transcription factor plate array assay kit was obtained fromSignosis Inc (USA). Briefly, 2×106 HeLa cells were treated with 100 nM41 and incubated for 6 hours before extracting the nuclear protein andcarrying out the TF plate array assay. The assay was carried outfollowing the manufacturer's protocol. In the case of each transcriptionfactor, the RLU value obtained for the cells treated with 41 wasdeducted from the respective values obtained for the untreated cells toobtain the differences in TF activation/inhibition.

Conjugation of Payload to Antibody

All ADC conjugations were completed using a similar methodology, anexample of which is provided below. 21.5 mg IgG1 antibody (8.0 mg/ml inPBS) were charged with EDTA to a final concentration of 2 mM. Reductionwas attained by adding 1.27 molar equivalents TCEP (10 mM in water) andincubating for 2 hours at 20° C. After 1.5 hours, a reduction in-processtest conjugation with Mal-vcMMAE was performed, and analyzed by HIC totest for the reduction level. As the target reduction level had not beenreached, another 0.1 molar equivalents TCEP were added and the reductiontime extended by 1 hour. After 0.5 hours, a second in-process test wasrun. After confirmation of the desired reduction level, 20% (v/v)Propylene glycol was added to the reduced antibody followed by 6.4 molarequivalents 141 (10 mM stock in DMSO). The solution was incubated for 1hour at rt. The reaction was quenched by adding 6.4 molar equivalentsN—Acetylcysteine (10 mM in water). The ADC was buffer exchanged via G25into PBS and washed by dead-end filtration (Vivaspin-20, 30 kDa MWCO,0.0006 m²) for 10 DVs. Samples were taken for analysis by HIC, SEC,PLRP, free toxin linker, Endosafe, and the concentration was determinedusing a SEC calibration curve. Aliquotting was carried out under laminarflow, and the product was stored at −80° C. Only disposable, sterile andpyrogen/DNA/RNA-free plasticware was used.

In Vivo Efficacy Studies

In vivo efficacy and tolerability studies were performed usingadaptations of the same basic protocol. An example of this is providedbelow:

Antitumour activity of the selected ADCs was assessed in tumourxenograft models (both cancer-derived and patient-derived) obtained byinoculation of the relevant cell-line (e.g., SK-BR-3 in the case ofADC1) in nude mice (CD-1 or appropriate depending on the cell-line).

Maximum tolerated dose (MTD) of the relevant ADC was established on 3-5CD1 mice (or equivalent) at four concentrations (e.g., 2.5 mg/kg, 5mg/kg, 7.5 mg/kg and 10 mg/kg) through IV administration once per weekfor a period of four weeks. Once the MTD was determined, an efficacystudy was initiated at doses under the maximum tolerated dose. Briefly,tumours were implanted onto the flank of the mice using a 23-gaugeneedle, and were randomly assigned to groups (e.g., control or ADC).After implantation, tumours were measured 3 times per week using digitalcalipers. The length and width of the tumour was measured and volumecalculated using the following formula: volume=(length×width²)/2. Thebodyweight of all mice on the study was measured and recorded 3 timesper week. Mice were observed daily and any signs of distress or changesto general condition (e.g., starred fur, lack of movement, difficultybreathing). Specific criteria were set for early termination, and thisonly occurred if tumour volume exceeded 1500 mm³, weight loss of ≥15%occurred or animals became compromised (e.g., inability to eat/drink).

Mice were housed in IVC cages (5 mice per cage) with individual miceidentified by ear punch. Cages, bedding and water were sanitized beforeuse. Animals were provided with Corn-o-cobs enrichment bedding toprovide environment enrichment and nesting material. All animals hadfree access to a standard certified commercial diet and water. Theanimal holding room was maintained as follows—room temperature at 20-24°C., humidity at 30-70% and a 12 h light/dark cycle used. Cages werechanged once a week with food and water replaced when necessary. Allprocedures were carried out under the guidelines of the Animal(Scientific Procedures) Act 1986.

All publications mentioned in the above specification are hereinincorporated by reference. Although illustrative embodiments of theinvention have been disclosed in detail herein, with reference to theaccompanying drawings, it is understood that the invention is notlimited to the precise embodiment and that various changes andmodifications can be effected therein by one skilled in the art withoutdeparting from the scope of the invention as defined by the appendedclaims and their equivalents.

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What is claimed:
 1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein: A is:

for each A1: (i) each Y₃ is independently NR₁₇, O, or S; (ii) each Y₄ isCH; and (iii) each Y₅ is independently CH, C(O)H, N, or S; or (i) eachY₃ is CH; (ii) each Y₄ is independently NR₁₇, O, or S; and (iii) each Y₅is independently CH, C(O)H, N, or S; for each A2: (i) each Y₆ isindependently CH or N; and (ii) each Y₇ is CH; or (i) each Y₆ is CH; and(ii) each Y₇ is independently CH or N; X₁ is —CR₁₃R₁₄—, —, CR₁₃R₁₄O—,—C(O)—, —C(O)NR₁₃—, —C(O)O—, —NR₁₃—, —NR₁₃C(O)—, —O—, —OC(O)—, or —S—; Lis an amino acid, a peptide chain having 2, 3, 4, 5, or 6 amino acids,—C₁₋₁₂ alkylene-, —(OCH₂)₁₋₁₂—, or —(OCH₂CH₂)₁₋₆—, wherein the C₁₋₁₂alkylene may optionally contain one or more carbon-carbon double bondsor carbon-carbon triple bonds, and further wherein the C₁₋₁₂ alkylene,—(OCH₂)₁₋₁₂—, and —(OCH₂CH₂)₁₋₆—may optionally and independently beinterrupted by one or more atoms or groups independently selected fromthe group consisting of —NH—, —O—, —S—, phenylene, and C₅₋₉heteroarylene; X₂ is absent; or X₂ is —CR₁₅R₁₆—, —CR₁₅R₁₆O—, —C(O)—,—C(O)NR₁₅—, —C(O)O—, —NR₁₅—, —NR₁₅C(O)—, —O—, —OC(O)—, or —S—; Y₁ is CHor N; Y₂ is CH or N; R₁ is R₇, (CH₂)_(m)—C(O)R₇, (CH₂)_(m)—C(O)NR₇R₈,(CH₂)_(m)—C(O)OR₇, (CH₂)_(m)—NR₇R₈, (CH₂)_(m)—OR₇, (CH₂)_(m)—S(O)₂R₇,═CH₂, ═CH—(CH₂)_(m)—CH₃, NHC(O)R₇, O—(CH₂)_(n)—C(O)NHR₇,O—(CH₂)_(n)−NR₇R₈, O—(CH₂)_(n)—NHC(O)R₇, OS(O)₂R₇, or ═O; R₂ is R₉,(CH₂)—C(O)R₉, (CH₂)—C(O)NR₉R₁₀, (CH₂)—C(O)OR₉, (CH₂)_(r)—NR₉R₁₀,(CH₂)_(r)—OR₉, (CH₂)_(r)—S(O)₂R₉, ═CH₂, ═CH—(CH₂)_(m)—CH₃, NHC(O)R₉,O—(CH₂)_(s)—C(O)NHR₉, O—(CH₂)_(s)—NR₉R₁₀, O—(CH₂)_(s)—NHC(O)R₉,OS(O)₂R₉, or ═O; R₃ is H, C₁₋₁₂ alkyl, or CH₂-phenyl; R₄ is phenyl orC₅₋₉ heteroaryl, each optionally substituted with one, two, or threesubstituents independently selected from the group consisting of C₁₋₆alkyl, (CH₂)_(j)—C(O)OR₁₁, (CH₂)_(j)—NR₁₁R₁₂, C(O)—NHR₂₄,C(O)—NH(CH₂)_(k)—C(NH)NR₁₁R₁₂, C(O)—NH(CH₂)_(k)—NR₁₁R₁₂, OH, OC₁₋₆alkyl, and O—(CH₂)_(k)—NR₁₁R₁₂; R₂₄ is phenyl, optionally substitutedwith one, two, or three substituents independently selected from thegroup consisting of C₁₋₆ alkyl, (CH₂)_(j)—C(O)OR₁₁, (CH₂)_(j)—NR₁₁R₁₂,C(O)—NHR₂₄, C(O)—NH(CH₂)_(k)—C(NH)NR₁₁R₁₂, C(O)—NH(CH₂)_(k)—NR₁₁R₁₂, OH,OC₁₋₆ alkyl, and O—(CH₂)_(k)—NR₁₁R₁₂; R₅ is H; and R₆ is OH or OC₁₋₆alkyl; or R₅ and R₆ together form a double bond; R₇ is H, C₁₋₁₂ alkyl,C₇₋₁₂ aralkyl, C₆₋₁₅ heteroarylalkyl, phenyl, or C₅₋₉ heteroaryl,wherein the aralkyl, heteroarylalkyl, phenyl, and heteroaryl are eachoptionally substituted with one, two, or three substituentsindependently selected from the group consisting of C₁₋₆ alkyl, OH, andOC₁₋₆ alkyl; R₈ is H or C₁₋₆ alkyl; R₉ is H, C₁₋₁₂ alkyl, C₇₋₁₂ aralkyl,C₆₋₁₅ heteroarylalkyl, phenyl, or C₅₋₉ heteroaryl, wherein the aralkyl,heteroarylalkyl, phenyl, and heteroaryl are each optionally substitutedwith one, two, or three substituents independently selected from thegroup consisting of C₁₋₆ alkyl, OH, and OC₁₋₆ alkyl; R₁₀ is H or C₁₋₆alkyl; R₁₁ is H or C₁₋₆ alkyl; R₁₂ is H or C₁₋₆ alkyl; R₁₃ is H or C₁₋₆alkyl; R₁₄ is H or C₁₋₆ alkyl; R₁₅ is H or C₁₋₆ alkyl; R₁₆ is H or C₁₋₆alkyl; R₁₇ is H or C₁₋₆ alkyl; R₁₉ is H or (CH₂)_(t)—NR₂₀R₂₁; R₂₀ is Hor C₁₋₆ alkyl; R₂₁ is H or C₁₋₆ alkyl; j is 0, 1, 2, 3, 4, 5, or 6; k is1, 2, 3, 4, 5, or 6; m is 0, 1, 2, 3, 4, 5, or 6; n is 1, 2, 3, 4, 5, or6; p is 0 or 1; q is 1, 2, 3, 4, 5, or 6; r is 0, 1, 2, 3, 4, 5, or 6; sis 1, 2, 3, 4, 5, or 6; t is 0, 1, 2, 3, 4, 5, or 6; and the dottedlines indicate the optional presence of a double bond between one ormore of C1 and C2, C2 and C3, and C3 and C4; with the provisos that: (1)R₄ is not optionally substituted indolyl; (2) at least one of Yi and Y₂is CH; and (3) when p is 0 and A is Al, then: (a) R₄ is not optionallysubstituted imidazolyl or optionally substituted pyrrolyl; (b) for atleast one Al, one of Y₃ and Y₄ is 0 or S; or (c) for at least one Al, Y₅is S.
 2. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein L is a peptide chain having 2, 3, 4, 5, or 6 aminoacids, —C₁₋₁₂ alkylene—, —(OCH₂)₁₋₁₂—, or —(OCH₂CH₂)₁₋₆—, wherein theC₁₋₁₂ alkylene may optionally contain one or more carbon-carbon doublebonds or carbon-carbon triple bonds, and further wherein the C₁₋₁₂alkylene, —(OCH₂)₁₋₁₂—, and —(OCH₂CH₂)₁₋₆—may optionally andindependently be interrupted by one or more atoms or groupsindependently selected from the group consisting of —NH—, —O—, —S—,phenylene, and C₅₋₉ heteroarylene.
 3. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R₁ is R₇,O—(CH₂)_(n)—C(O)NHR₇, or O—(CH₂)_(n)—NHC(O)R₇.
 4. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein R₂ is H. 5.The compound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R₃ is CH₃ or CH₂CH₃.
 6. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein: R₄ is phenyl,pyrrolyl, imidazolyl, furanyl, thiophenyl, oxazolyl, thiazolyl, pyridyl,benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, orbenzothiazolyl, each optionally substituted with one, two, or threesubstituents independently selected from the group consisting of C₁₋₆alkyl, (CH₂)_(j)—C(O)OR₁₁, (CH₂)_(j)—N₁₁R₁₂, C(O)—NHR₂₄,C(O)—NH(CH₂)_(k)—C(NH)NR₁₁R₁₂, C(O)—NH(CH₂)_(k)—NR₁₁R₁₂, OH, OC₁₋₆alkyl, and O—(CH₂)_(k)—NR₁₁R₁₂; R₂₄ is phenyl, substituted with one(CH₂)_(j)—R₁₈; and R₁₈ is C(O)OR₁₁ or NR₁₁R₁₂.
 7. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein: R₄ isselected from the group consisting of:

Z₁ is —NH—, —N(CH₃)—, —O—, or —S—; Z₂ is CH or N; Z₃ is —O— or —S—; Z₄is CH or N; R₂₂ is (CH₂)_(j)—C(O)OR₁₁, (CH₂)_(j)—NR₁₁R₁₂, or C(O)—NHR₂₄;R₂₃ is H or C₁₋₆ alkyl; R₂₄ is phenyl, substituted with one(CH₂)_(j)—R₁₈; and R₁₈ is C(O)OR₁₁ or NR₁₁R₁₂.
 8. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein R₅ and R₆together form a double bond.
 9. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein: R₂₄ is phenyl,substituted with one (CH₂)_(j)—R₁₈; and R₁₈ is C(O)OR₁₁ or NR₁₁R₁₂. 10.The compound of claim 1, or a pharmaceutically acceptable salt thereof,wherein p is
 1. 11. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein: A is:

for each A1: each Y₃ is N(CH₃); each Y₄ is CH; for each A2: each Y₆ isCH; and each Y₇ is CH; X₁ is —O—; L is —C₁₋₁₂ alkylene—, optionallyinterrupted by one or more atoms or groups independently selected fromthe group consisting of phenylene and C₅₋₉ heteroarylene; X₂ is—C(O)NH—; R₁ is H; R₂ is H; R₃ is CH₃; and the dotted lines indicatingthe optional presence of a double bond between one or more of C1 and C2,C2 and C3, and C3 and C4 are absent.
 12. The compound of claim 11, or apharmaceutically acceptable salt thereof, wherein R₄ is phenyl, furanyl,thiophenyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl, benzothiophenyl,benzimidazolyl, benzoxazolyl, or benzothiazolyl, each optionallysubstituted with one, two, or three substituents independently selectedfrom the group consisting of C₁₋₆ alkyl, (CH₂)_(j)—C(O)OR₁₁,(CH₂)_(j)—NR₁₁R₁₂, C(O)—NHR₂₄, C(O)—NH(CH₂)_(k)—C(NH)NR₁₁R₁₂,C(O)—NH(CH₂)_(k)—NR₁₁R₁₂, OH, OC₁₋₆ alkyl, and O—(CH₂)_(k)—NR₁₁R₁₂. 13.The compound of claim 11, or a pharmaceutically acceptable salt thereof,wherein R₄ is phenyl, thiophenyl, oxazolyl, thiazolyl, benzothiophenyl,or benzothiazolyl, each optionally substituted with one or twosubstituents independently selected from the group consisting of C₁₋₆alkyl, (CH₂)_(j)—C(O)OR₁₁, (CH₂)_(j)—NR₁₁R₁₂, C(O)—NHR₂₄,C(O)—NH(CH₂)_(k)—C(NH)NR₁₁R₁₂, C(O)—NH(CH₂)_(k)—NR₁₁R₁₂, OH, OC₁₋₆alkyl, and O—(CH₂)_(k)—NR₁₁R₁₂.
 14. The compound of claim 1, wherein thecompound is of formula (XV):

or a pharmaceutically acceptable salt thereof.
 15. The compound of claim1, wherein the compound is of formula (XVI), formula (XVII), formula(XVIII), or formula (XIX):

or a pharmaceutically acceptable salt thereof, wherein: L is —C₁₋₁₂alkylene—; each Y₅ is independently CH or N; Z₁ is —NH—, —N(CH₃)—, —O—,or —S—; Z₂ is CH or N; Z₃ is —O— or —S—; Z₄ is CH or N; R₂₂ is(CH₂)_(j)—C(O)OH, (CH₂)_(j)—C(O)OC₁₋₆ alkyl, (CH₂)_(j)—NR₁₁R₁₂, orC(O)—NHR₂₄; R₂₃ is H or C₁₋₆ alkyl; R₂₄ is phenyl, substituted with one(CH₂)_(j)—R₁₈; and R₁₈ is C(O)OR₁₁ or NR₁₁R₁₂; with the proviso thatwhen the compound is of formula (XVI) and p is 0, then Z₁ is —O— or —S—.16. The compound of claim 2, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 17. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable excipient,carrier, or diluent.
 18. A compound selected from the group consistingof:

or a pharmaceutically acceptable salt thereof.
 19. A compound selectedfrom the group consisting of:

or a pharmaceutically acceptable salt thereof.
 20. A compound selectedfrom the group consisting of:

or a pharmaceutically acceptable salt thereof.